Geometric control of ciliated band regulatory states in the sea urchin embryo.

The trapezoidal ciliated band (CB) of the postgastrular sea urchin embryo surrounds the oral ectoderm, separating it from adjacent embryonic territories. Once differentiated, the CB is composed of densely arranged cells bearing long cilia that endow the larva with locomotion and feeding capability. The spatial pattern from which the CB will arise is first evidenced during pregastrular stages by expression of the pioneer gene onecut. Immediately after gastrulation, the CB consists of four separate regulatory state domains, each of which expresses a unique set of transcription factors: (1) the oral apical CB, located within the apical neurogenic field; (2) the animal lateral CB, which bilaterally separates the oral from aboral ectoderm; (3) the vegetal lateral CB, which bilaterally serves as signaling centers; and (4) the vegetal oral CB, which delineates the boundary with the underlying endoderm. Remarkably, almost all of the regulatory genes specifically expressed within these domains are downregulated by interference with SoxB1 expression, implying their common activation by this factor. Here, we show how the boundaries of the CB subdomains are established, and thus ascertain the design principle by which the geometry of this unique and complex regulatory state pattern is genomically controlled. Each of these boundaries, on either side of the CB, is defined by spatially confined transcriptional repressors, the products of regulatory genes operating across the border of each subdomain. In total this requires deployment of about ten different repressors, which we identify in this work, thus exemplifying the complexity of information required for spatial regulatory organization during embryogenesis.

Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo.

Wnt signaling affects cell-fate specification processes throughout embryonic development. Here we take advantage of the well-studied gene regulatory networks (GRNs) that control pregastrular sea urchin embryogenesis to reveal the gene regulatory functions of the entire Wnt-signaling system. Five wnt genes, three frizzled genes, two secreted frizzled-related protein 1 genes, and two Dickkopf genes are expressed in dynamic spatial patterns in the pregastrular embryo of Strongylocentrotus purpuratus. We present a comprehensive analysis of these genes in each embryonic domain. Total functions of the Wnt-signaling system in regulatory gene expression throughout the embryo were studied by use of the Porcupine inhibitor C59, which interferes with zygotic Wnt ligand secretion. Morpholino-mediated knockdown of each expressed Wnt ligand demonstrated that individual Wnt ligands are functionally distinct, despite their partially overlapping spatial expression. They target specific embryonic domains and affect particular regulatory genes. The sum of the effects of blocking expression of individual wnt genes is shown to equal C59 effects. Remarkably, zygotic Wnt-signaling inputs are required for only three general aspects of embryonic specification: the broad activation of endodermal GRNs, the regional specification of the immediately adjacent stripe of ectoderm, and the restriction of the apical neurogenic domain. All Wnt signaling in this pregastrular embryo is short range (and/or autocrine). Furthermore, we show that the transcriptional drivers of wnt genes execute important specification functions in the embryonic domains targeted by the ligands, thus connecting the expression and function of wnt genes by encoded cross-regulatory interactions within the specific regional GRNs.

Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo.

By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo.

New regulatory circuit controlling spatial and temporal gene expression in the sea urchin embryo oral ectoderm GRN.

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.

Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos.

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.

Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown.

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-ß 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 lockdown the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo.

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 occur within this territory which depend 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 among 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.

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 among 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.

Protein-coding gene promoters in Methanocaldococcus (Methanococcus) jannaschii.

Although Methanocaldococcus (Methanococcus) jannaschii was the first archaeon to have its genome sequenced, little is known about the promoters of its protein-coding genes. To expand our knowledge, we have experimentally identified 131 promoters for 107 protein-coding genes in this genome by mapping their transcription start sites. Compared to previously identified promoters, more than half of which are from genes for stable RNAs, the protein-coding gene promoters are qualitatively similar in overall sequence pattern, but statistically different at several positions due to greater variation among their sequences. Relative binding affinity for general transcription factors was measured for 12 of these promoters by competition electrophoretic mobility shift assays. These promoters bind the factors less tightly than do most tRNA gene promoters. When a position weight matrix (PWM) was constructed from the protein gene promoters, factor binding affinities correlated with corresponding promoter PWM scores. We show that the PWM based on our data more accurately predicts promoters in the genome and transcription start sites than could be done with the previously available data. We also introduce a PWM logo, which visually displays the implications of observing a given base at a position in a sequence.

A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo.

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 24 h 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.

A whole-genome approach to identifying protein binding sites: promoters in Methanocaldococcus (Methanococcus) jannaschii.

We have adapted an electrophoretic mobility shift assay (EMSA) to isolate genomic DNA fragments that bind the archaeal transcription initiation factors TATA-binding protein (TBP) and transcription factor B (TFB) to perform a genome-wide search for promoters. Mobility-shifted fragments were cloned, tested for their ability to compete with known promoter-containing fragments for a limited concentration of transcription factors, and sequenced. We applied the method to search for promoters in the genome of Methanocaldococcus jannaschii. Selection was most efficient for promoters of tRNA genes and genes for several presumed small non-coding RNAs (ncRNA). Protein-coding gene promoters were dramatically underrepresented relative to their frequency in the genome. The repeated isolation of these genomic regions was partially rectified by including a hybridization-based screening. Sequence alignment of the affinity-selected promoters revealed previously identified TATA box, BRE, and the putative initiator element. In addition, the conserved bases immediately upstream and downstream of the BRE and TATA box suggest that the composition and structure of archaeal natural promoters are more complicated.