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1.  Quantitative developmental transcriptomes of the sea urchin Strongylocentrotus purpuratus 
Developmental biology  2013;385(2):160-167.
Development depends on the precise control of gene expression in time and space. A critical step towards understanding the global gene regulatory networks underlying development is to obtain comprehensive information on gene expression. In this study, we measured expression profiles for the entire expressed gene set during sea urchin embryonic development. We confirmed the reliability of these profiles by comparison with NanoString measurements for a subset of genes and with literature values. The data show that ~16,500 genes have been activated by the end of embryogenesis, and for half of them the transcript abundance changes more than 10-fold during development. From this genome scale expression survey, we show that complex patterns of expression by many genes underlie embryonic development, particularly during the early stages before gastrulation. An intuitive web application for data query and visualization is presented to facilitate use of this large dataset.
doi:10.1016/j.ydbio.2013.11.019
PMCID: PMC3898891  PMID: 24291147
sea urchin; developmental transcriptome; RNA sequencing
2.  Genomic control of patterning 
The development of multicellular organisms involves the partitioning of the organism into territories of cells of specific structure and function. The information for spatial patterning processes is directly encoded in the genome. The genome determines its own usage depending on stage and position, by means of interactions that constitute gene regulatory networks (GRNs). The GRN driving endomesoderm development in sea urchin embryos illustrates different regulatory strategies by which developmental programs are initiated, orchestrated, stabilized or excluded to define the pattern of specified territories in the developing embryo.
doi:10.1387/ijdb.072495ip
PMCID: PMC3967875  PMID: 19378258
gene regulatory network; cis-regulatory module; network subcircuit; sea urchin; development
3.  NEW REGULATORY CIRCUIT CONTROLLING SPATIAL AND TEMPORAL GENE EXPRESSION IN THE SEA URCHIN EMBRYO ORAL ECTODERM GRN 
Developmental biology  2013;382(1):268-279.
The sea urchin oral ectoderm gene regulatory network (GRN) model has increased in complexity as additional genes are added to it, revealing its multiple spatial regulatory state domains. The formation of the oral ectoderm begins with an oral-aboral redox gradient, which is interpreted by the cis-regulatory system of the nodal gene to cause its expression on the oral side of the embryo. Nodal signaling drives cohorts of regulatory genes within the oral ectoderm and its derived subdomains. Activation of these genes occurs sequentially, spanning the entire blastula stage. During this process the stomodeal subdomain emerges inside of the oral ectoderm, and bilateral subdomains defining the lateral portions of the future ciliary band emerge adjacent to the central oral ectoderm. Here we examine two regulatory genes encoding repressors, sip1 and ets4, which selectively prevent transcription of oral ectoderm genes until their expression is cleared from the oral ectoderm as an indirect consequence of Nodal signaling. We show that the timing of transcriptional de-repression of sip1 and ets4 targets which occurs upon their clearance explains the dynamics of oral ectoderm gene expression. In addition two other repressors, the direct Nodal target not, and the feed forward Nodal target goosecoid, repress expression of regulatory genes in the central animal oral ectoderm thereby confining their expression to the lateral domains of the animal ectoderm. These results have permitted construction of an enhanced animal ectoderm GRN model highlighting the repressive interactions providing precise temporal and spatial control of regulatory gene expression.
doi:10.1016/j.ydbio.2013.07.027
PMCID: PMC3783610  PMID: 23933172
Sea urchin; oral ectoderm; sip1; ets4; gene regulatory network
4.  Building Developmental Gene Regulatory Networks 
Animal development is an elaborate process programmed by genomic regulatory instructions. Regulatory genes encode transcription factors and signal molecules, and their expression is under the control of cis-regulatory modules that define the logic of transcriptional responses to the inputs of other regulatory genes. The functional linkages amongst regulatory genes constitute the gene regulatory networks (GRNs) that govern cell specification and patterning in development. Constructing such networks requires identification of the regulatory genes involved and characterization of their temporal and spatial expression patterns. Interactions (activation/repression) among transcription factors or signals can be investigated by large-scale perturbation analysis, in which the function of each gene is specifically blocked. Resultant expression changes are then integrated to identify direct linkages, and to reveal the structure of the GRN. Predicted GRN linkages can be tested and verified by cis-regulatory analysis. The explanatory power of the GRN was shown in the lineage specification of sea urchin endomesoderm. Acquiring such networks is essential for a systematic and mechanistic understanding of the developmental process.
doi:10.1002/bdrc.20152
PMCID: PMC2747644  PMID: 19530131
gene regulatory network; development; cis-regulatory analysis; sea urchin
5.  Modeling the dynamics of transcriptional gene regulatory networks for animal development 
Developmental biology  2008;325(2):317-328.
The dynamic process of cell fate specification is regulated by networks of regulatory genes. The architecture of the network defines the temporal order of specification events. To understand the dynamic control of the developmental process, the kinetics of mRNA and protein synthesis and the response of the cis-regulatory modules to transcription factor concentration must be considered. Here we review mathematical models for mRNA and protein synthesis kinetics which are based on experimental measurements of the rates of the relevant processes. The model comprises the response functions of cis-regulatory modules to their transcription factor inputs, by incorporating binding site occupancy and its dependence on biologically measurable quantities. We use this model to simulate gene expression, to distinguish between cis-regulatory execution of “AND” and “OR” logic functions, rationalize the oscillatory behavior of certain transcriptional auto-repressors and to show how linked subcircuits can be dealt with. Model simulations display the effects of mutation of binding sites, or perturbation of upstream gene expression. The model is a generally useful tool for understanding gene regulation and the dynamics of cell fate specification.
doi:10.1016/j.ydbio.2008.10.043
PMCID: PMC4100934  PMID: 19028486
Mathematical modeling; Gene regulation in development; Dynamics; Logic functions
6.  A New Software Package for Predictive Gene Regulatory Network Modeling and Redesign 
Journal of Computational Biology  2013;20(6):419-423.
Abstract
The efficacy of a newly created software package for predictive modeling of developmental gene regulatory networks (GRNs) has recently been demonstrated (Peter et al., 2012). The program GeNeTool computes spatial gene expression patterns based on GRN interactions and thereby allows the direct comparison of predicted and observed spatial expression patterns. GeNeTool also permits in silico exploration of both cis- and trans- perturbations of GRN interactions. Here, we present this program, review briefly its major features and applications, and provide a detailed and accessible tutorial.
doi:10.1089/cmb.2012.0297
PMCID: PMC3667423  PMID: 23614576
artificial life; gene networks; genetic analysis; recognition of genes; regulatory elements
7.  Logic of gene regulatory networks 
Current opinion in biotechnology  2007;18(4):351-354.
Regulatory networks of transcription factors and signaling molecules lie at the heart of development. Their architecture implements logic functions whose execution propels cells from one regulatory state to the next, thus driving development forward. As an example of a subcircuit that translates transcriptional input into developmental output we consider a particularly simple case, the regulatory processes underlying pigment cell formation in sea urchin embryos. The regulatory events in this process can be represented as elementary logic functions.
doi:10.1016/j.copbio.2007.07.008
PMCID: PMC2031216  PMID: 17689240
8.  Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network 
Developmental biology  2007;306(2):860-869.
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.
doi:10.1016/j.ydbio.2007.03.033
PMCID: PMC2063469  PMID: 17451671
Nodal; Oral ectoderm; Gene regulatory network; Community effect; TGF-beta; bZIP; SMAD; Sea urchin; Positive feedback regulation; cis-regulatory analysis
9.  The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage☆ 
Developmental biology  2009;340(2):188-199.
As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.
doi:10.1016/j.ydbio.2009.10.037
PMCID: PMC3981691  PMID: 19895806
Gene regulatory network; Endoderm; Mesoderm; Embryonic specification
10.  A gene regulatory network controlling the embryonic specification of endoderm 
Nature  2011;474(7353):635-639.
Specification of endoderm is the prerequisite for gut formation in the embryogenesis of bilaterian organisms. Modern lineage labelling studies 1–3 have shown that in the sea urchin embryo model system, descendants of the veg1 and veg2 cell lineages produce the endoderm, and that the veg2 lineage also gives rise to mesodermal cell types. It is known that Wnt/β-catenin signalling is required for endoderm specification4–6 and Delta/Notch signalling is required for mesoderm specification7–9. Some direct cis-regulatory targets of these signals have been found10,11 and various phenomenological patterns of gene expression have been observed in the pre-gastrular endomesoderm. However, no comprehensive, causal explanation of endoderm specification has been conceived for sea urchins, nor for any other deuterostome. Here we propose a model, on the basis of the underlying genomic control system, that provides such an explanation, built at several levels of biological organization. The hardwired core of the control system consists of the cis-regulatory apparatus of endodermal regulatory genes, which determine the relationship between the inputs to which these genes are exposed and their outputs. The architecture of the network circuitry controlling the dynamic process of endoderm specification then explains, at the system level, a sequence of developmental logic operations, which generate the biological process. The control system initiates noninteracting endodermal and mesodermal gene regulatory networks in veg2-derived cells and extinguishes the endodermal gene regulatory network in mesodermal precursors. It also generates a cross-regulatory network that specifies future anterior endoderm in veg2 descendants and institutes a distinct network specifying posterior endoderm in veg1-derived cells. The network model provides an explanatory framework that relates endoderm specification to the genomic regulatory code.
doi:10.1038/nature10100
PMCID: PMC3976212  PMID: 21623371
11.  Emerging properties of animal gene regulatory networks 
Nature  2010;468(7326):911-920.
Gene regulatory networks (GRNs) provide system level explanations of developmental and physiological functions in the terms of the genomic regulatory code. Depending on their developmental functions, GRNs differ in their degree of hierarchy, and also in the types of modular sub-circuit of which they are composed, although there is a commonly employed sub-circuit repertoire. Mathematical modelling of some types of GRN sub-circuit has deepened biological understanding of the functions they mediate. The structural organization of various kinds of GRN reflects their roles in the life process, and causally illuminates both developmental and evolutionary process.
doi:10.1038/nature09645
PMCID: PMC3967874  PMID: 21164479
12.  Diversification of Oral and Aboral Mesodermal Regulatory States in Pregastrular Sea Urchin Embryos 
Developmental biology  2012;375(1):92-104.
Summary
Specification of the non-skeletogenic mesoderm (NSM) in sea urchin embryos depends on Delta signaling. Signal reception leads to expression of regulatory genes that later contribute to the aboral NSM regulatory state. In oral NSM, this is replaced by a distinct oral regulatory state in consequence of Nodal signaling. Through regulome wide analysis we identify the homeobox gene not as an immediate Nodal target. not expression in NSM causes extinction of the aboral regulatory state in the oral NSM, and expression of a new suite of regulatory genes. All NSM specific regulatory genes are henceforth expressed exclusively, in oral or aboral domains, presaging the mesodermal cell types that will emerge. We have analyzed the regulatory linkages within the aboral NSM gene regulatory network. A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state.
doi:10.1016/j.ydbio.2012.11.033
PMCID: PMC3570723  PMID: 23261933
mesoderm; nodal signaling; oral/aboral axis; not; gcm; gataE; gene regulatory network
13.  A new method, using cis-regulatory control, for blocking embryonic gene expression 
Developmental biology  2008;318(2):360-365.
Many genes, and particularly regulatory genes, are utilized multiple times in unrelated phases of development. For studies of gene function during embryogenesis there is often need of a method for interfering with expression only at a specific developmental time or place. Here we show that in sea urchin embryos cis-regulatory control systems which operate only at specific times and places can be used to drive expression of short designed sequences targeting given primary transcripts, thereby effectively taking out the function of the target genes. The active sequences are designed to be complementary to intronic sequences of the primary transcript of the target genes. In this work the target genes were the transcription factors alx1 and ets1, both required for skeletogenesis, and the regulatory drivers were from the sm30 and tbr genes. The sm30 gene is expressed only after skeletogenic cell ingression. When its regulatory apparatus was used as driver, the alx1 and ets1 repression constructs had the effect of preventing postgastrular skeletogenesis, while not interfering with earlier alx1 and ets1 function in promoting skeletogenic mesenchyme ingression. In contrast repression constructs using the tbr driver, which is active in blastula stage, block ingression. This method thus provides the opportunity to study regulatory requirements of skeletogenesis after ingression, and may be similarly useful in many other developmental contexts.
doi:10.1016/j.ydbio.2008.02.056
PMCID: PMC3929273  PMID: 18423438
Sea urchin embryo; Skeletogenic mesenchyme; Intron antisense RNA; Gene knockdown
14.  Gene Regulatory Control in the Sea Urchin Aboral Ectoderm: Spatial Initiation, Signaling Inputs, and Cell Fate Lockdown 
Developmental biology  2012;374(1):245-254.
The regulation of oral-aboral ectoderm specification in the sea urchin embryo has been extensively studied in recent years. The oral-aboral polarity is initially imposed downstream of a redox gradient induced by asymmetric maternal distribution of mitochondria. Two TGF-β signaling pathways, Nodal and BMP, are then respectively utilized in the generation of oral and aboral regulatory states. However, a causal understanding of the regulation of aboral ectoderm specification has been lacking. In this work control of aboral ectoderm regulatory state specification was revealed by combining detailed regulatory gene expression studies, perturbation and cis-regulatory analyses. Our analysis illuminates a dynamic system where different factors dominate at different developmental times. We found that the initial activation of aboral genes depends directly on the redox sensitive transcription factor, hypoxia inducible factor 1α (HIF-1α). Two BMP ligands, BMP2/4 and BMP5/8, then significantly enhance aboral regulatory gene transcription. Ultimately, encoded feedback wiring lock-down the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.
doi:10.1016/j.ydbio.2012.11.013
PMCID: PMC3548969  PMID: 23211652
gene regulatory networks; cis-regulatory analysis; ectoderm specification; sea urchin; developmental control
15.  Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo 
Developmental biology  2012;369(2):377-385.
The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occurs within this territory which depends directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations amongst the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.
doi:10.1016/j.ydbio.2012.06.022
PMCID: PMC3423475  PMID: 22771578
nodal signaling; gene regulatory network; oral ectoderm specification
16.  Cis-regulatory logic driving glial cells missing: self-sustaining circuitry in later embryogenesis 
Developmental biology  2012;364(2):259-267.
The glial cells missing (gcm) regulatory gene of the sea urchin Strongylocentrotus purpuratus is first expressed in veg2 daughter cells as the genomic target of late cleavage stage Delta-Notch signaling from the skeletogenic mesoderm precursors. Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. Here we report on a later acting cis-regulatory module that assumes control of gcm expression by the early mesenchyme blastula stage and maintains it through pigment cell differentiation and dispersal. Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. Significantly, six1 mRNA localizes to gcm+ cells from the mesenchyme blastula stage onwards. Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. This often employed cross regulatory GRN feature here ensures self-sustaining gcm output in a cohort of fully specified pigment cell precursors at a relatively early developmental stage.
PMCID: PMC3561781  PMID: 22509525
echinoid; pigment cell; mesoderm; cis-regulation; autoregulatory; intergenic feedback loop
17.  A comprehensive analysis of Delta signaling in pre-gastrular sea urchin embryos 
Developmental Biology  2012;364(1):77-87.
In sea urchin embryos Delta signaling specifies non-skeletogenic mesoderm (NSM). Despite the identification of some direct targets, several aspects of Delta Notch (D/N) signaling remain supported only by circumstantial evidence. To obtain a detailed and more complete image of Delta function we followed a systems biology approach and evaluated the effects of D/N perturbation on expression levels of 205 genes up to gastrulation. This gene set includes virtually all transcription factors that are expressed in a localized fashion by mid-gastrulation, and which thus provide spatial regulatory information to the embryo. Also included are signaling factors and some pigment cell differentiation genes. We show that the number of pregastrular D/N signaling targets among these regulatory genes is small and is almost exclusively restricted to non-skeletogenic mesoderm genes. However, Delta signaling also activates foxY in the small micromeres. As is the early NSM, the small micromeres are in direct contact with Delta expressing skeletogenic mesoderm. In contrast, no endoderm regulatory genes are activated by Delta signaling even during the second phase of delta expression, when this gene is transcribed in NSM cells adjacent to the endoderm. During this phase Delta provides an ongoing input which continues to activate foxY expression in small micromere progeny. Disruption of the second phase of Delta expression specifically abolishes specification of late mesodermal derivatives such as the coelomic pouches to which the small micromeres contribute.
doi:10.1016/j.ydbio.2012.01.017
PMCID: PMC3294105  PMID: 22306924
Delta; Notch; DAPT; FoxY; Mesoderm; Coelomic Pouch; Pigment; Small Micromeres; Sea Urchin
18.  Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus 
Developmental biology  2011;357(2):505-517.
Deployment of the gene regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an autorepressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.
doi:10.1016/j.ydbio.2011.06.016
PMCID: PMC3164750  PMID: 21723273
Alx1 gene; cis-regulation; skeletogenic micromere lineage; gene regulatory network; tagged reporter assay
19.  EVOLUTIONARY BIOSCIENCE AS REGULATORY SYSTEMS BIOLOGY 
Developmental biology  2011;357(1):35-40.
At present several entirely different explanatory approaches compete to illuminate the mechanisms by which animal body plans have evolved. Their respective relevance is briefly considered here in the light of modern knowledge of genomes and the regulatory processes by which development is controlled. Just as development is a system property of the regulatory genome, so causal explanation of evolutionary change in developmental process must be considered at a system level. Here I enumerate some mechanistic consequences that follow from the conclusion that evolution of the body plan has occurred by alteration of the structure of developmental gene regulatory networks. The hierarchy and multiple additional design features of these networks act to produce Boolean regulatory state specification functions at upstream phases of development of the body plan. These are created by the logic outputs of network subcircuits, and in modern animals these outputs are impervious to continuous adaptive variation unlike genes operating more peripherally in the network.
doi:10.1016/j.ydbio.2011.02.004
PMCID: PMC3135751  PMID: 21320483
GRN evolution; GRN selection
20.  NETWORK DESIGN PRINCIPLES FROM THE SEA URCHIN EMBRYO 
As gene regulatory network models encompass more and more of the specification processes underlying sea urchin embryonic development, topological themes emerge that imply the existence of structural network “building blocks”. These are subcircuits which perform given logic operations in the spatial control of gene expression. The various parts of the sea urchin gene regulatory networks offer instances of the same subcircuit topologies accomplishing the same developmental logic functions but using different genes. These subcircuits are dedicated to specific developmental functions, unlike simpler “motifs”, and may indicate a repertoire of specific devices of which developmental gene regulatory networks are composed.
doi:10.1016/j.gde.2009.10.007
PMCID: PMC3428372  PMID: 19913405
21.  Barcoded DNA-Tag Reporters for Multiplex Cis-Regulatory Analysis 
PLoS ONE  2012;7(4):e35934.
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.
doi:10.1371/journal.pone.0035934
PMCID: PMC3339872  PMID: 22563420
22.  Evolution of Gene Regulatory Networks that Control Embryonic Development of the Body Plan 
Cell  2011;144(6):970-985.
SUMMARY
Alteration of the functional organization of the gene regulatory networks (GRNs) that control development of the body plan causes evolutionary change in animal morphology. A major mechanism of evolutionary change in GRN structure is alteration of cis-regulatory modules that determine regulatory gene expression. Both evolutionary conservation and evolutionary innovation must be considered in terms of GRN structure. Here we consider the causes and consequences of GRN evolution, both from an a priori point of view, and in light of extensive recent research on developmental regulatory alterations occurring at different levels of GRN hierarchy. Some GRN subcircuits are of great antiquity while other aspects are highly flexible and thus in any given genome more recent. Both evolutionary conservation and evolutionary innovation occur at the level of whole GRN subcircuits. This mosaic view of the evolution of GRN structure explains major aspects of evolutionary process, such as hierarchical phylogeny and discontinuities of paleontological change and stasis.
doi:10.1016/j.cell.2011.02.017
PMCID: PMC3076009  PMID: 21414487
23.  High accuracy, high-resolution prevalence measurement for the majority of locally expressed regulatory genes in early sea urchin development 
Gene expression patterns : GEP  2010;10(4-5):177-184.
Accurate measurements of transcript abundance are a prerequisite to understand gene activity in development. Using the NanoString nCounter, an RNA counting device, we measured the prevalence of 172 transcription factors and signaling molecules in early sea urchin development. These measurements show high fidelity over more than five orders of magnitude down to a few transcripts per embryo. Most of the genes included are locally restricted in their spatial expression, and contribute to the divergent regulatory states of cells in the developing embryo. In order to obtain high-resolution expression, profiles from fertilization to late gastrulation samples were collected at hourly intervals. The measured time courses agree well with, and substantially extend, prior relative abundance measurements obtained by quantitative PCR. High temporal resolution permits sequences of successively activated genes to be precisely delineated providing an ancillary tool for assembling maps of gene regulatory networks. The data are available via an interactive website for quick plotting of selected time courses.
doi:10.1016/j.gep.2010.04.002
PMCID: PMC2902461  PMID: 20398801
Transcription factor; Gene expression time course; mRNA prevalence measurement; Embryogenesis
24.  The gene regulatory network basis of the “community effect,” and analysis of a sea urchin embryo example 
Developmental biology  2009;340(2):170-178.
The “Community Effect” denotes intra-territorial signaling amongst cells which constitute a particular tissue or embryonic progenitor field. The cells of the territory express the same transcriptional regulatory state, and the intra-territorial signaling is essential to maintenance of this specific regulatory state. The structure of the underlying gene regulatory network (GRN) subcircuitry explains the genomically wired mechanism by which community effect signaling is linked to the continuing transcriptional generation of the territorial regulatory state. A clear example is afforded by the oral ectoderm GRN of the sea urchin embryo where cis-regulatory evidence, experimental embryology, and network analysis combine to provide a complete picture. We review this example and consider less well known but similar cases in other developing systems where the same subcircuit GRN topology is present. To resolve mechanistic issues that arise in considering how community effect signaling could operate to produce its observed effects, we construct and analyze the behavior of a quantitative model of community effect signaling in the sea urchin embryo oral ectoderm. Community effect network topology could constitute part of the genomic regulatory code that defines transcriptional function in multicellular tissues composed of cells in contact, and hence may have arisen as a metazoan developmental strategy.
doi:10.1016/j.ydbio.2009.06.007
PMCID: PMC2854306  PMID: 19523466
Community effect; Intradomain signaling; sea urchin embryo; nodal gene regulation
25.  Modularity and design principles in the sea urchin embryo gene regulatory network 
FEBS letters  2009;583(24):3948-3958.
The gene regulatory network (GRN) established experimentally for the pre-gastrular sea urchin embryo provides causal explanations of the biological functions required for spatial specification of embryonic regulatory states. Here we focus on the structure of the GRN which controls the progressive increase in complexity of territorial regulatory states during embryogenesis; and on the types of modular subcircuits of which the GRN is composed. Each of these subcircuit topologies executes a particular operation of spatial information processing. The GRN architecture reflects the particular mode of embryogenesis represented by sea urchin development. Network structure not only specifies the linkages constituting the genomic regulatory code for development, but also indicates the various regulatory requirements of regional developmental processes.
doi:10.1016/j.febslet.2009.11.060
PMCID: PMC2810318  PMID: 19932099
Subcircuit structure/function; Spatial transcriptional regulation; Embryonic specification

Results 1-25 (34)