Endomesoderm Gene Regulatory Network Research Articles

Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition

Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.

Conserved regulatory state expression controlled by divergent developmental gene regulatory networks in echinoids.

Evolution of the animal body plan is driven by changes in developmental gene regulatory networks (GRNs), but how networks change to control novel developmental phenotypes remains, in most cases, unresolved. Here, we address GRN evolution by comparing the endomesoderm GRN in two echinoid sea urchins, Strongylocentrotus purpuratus and Eucidaris tribuloides, with at least 268 million years of independent evolution. We first analyzed the expression of twelve transcription factors and signaling molecules of the S. purpuratus GRN in E. tribuloides embryos, showing that orthologous regulatory genes are expressed in corresponding endomesodermal cell fates in the two species. However, perturbation of regulatory genes revealed that important regulatory circuits of the S. purpuratus GRN are significantly different in E. tribuloides For example, mesodermal Delta/Notch signaling controls exclusion of alternative cell fates in E. tribuloides but controls mesoderm induction and activation of a positive feedback circuit in S. purpuratus These results indicate that the architecture of the sea urchin endomesoderm GRN evolved by extensive gain and loss of regulatory interactions between a conserved set of regulatory factors that control endomesodermal cell fate specification.

Assessing regulatory information in developmental gene regulatory networks

Gene regulatory networks (GRNs) provide a transformation function between the static genomic sequence and the primary spatial specification processes operating development. The regulatory information encompassed in developmental GRNs thus goes far beyond the control of individual genes. We here address regulatory information at different levels of network organization, from single node to subcircuit to large-scale GRNs and discuss how regulatory design features such as network architecture, hierarchical organization, and cis-regulatory logic contribute to the developmental function of network circuits. Using specific subcircuits from the sea urchin endomesoderm GRN, for which both circuit design and biological function have been described, we evaluate by Boolean modeling and in silico perturbations the import of given circuit features on developmental function. The examples include subcircuits encoding positive feedback, mutual repression, and coherent feedforward, as well as signaling interaction circuitry. Within the hierarchy of the endomesoderm GRN, these subcircuits are organized in an intertwined and overlapping manner. Thus, we begin to see how regulatory information encoded at individual nodes is integrated at all levels of network organization to control developmental process.

Synthetic in vivo validation of gene network circuitry

Embryonic development is controlled by networks of interacting regulatory genes. The individual linkages of gene regulatory networks (GRNs) are customarily validated by functional cis-regulatory analysis, but an additional approach to validation is to rewire GRN circuitry to test experimentally predictions derived from network structure. Here we use this synthetic method to challenge specific predictions of the sea urchin embryo endomesoderm GRN. Expression vectors generated by in vitro recombination of exogenous sequences into BACs were used to cause elements of a nonskeletogenic mesoderm GRN to be deployed in skeletogenic cells and to detect their effects. The result of reengineering the regulatory circuitry in this way was to divert the developmental program of these cells from skeletogenesis to pigment cell formation, confirming a direct prediction of the GRN. In addition, the experiment revealed previously undetected cryptic repression functions.

Wnt6 activates endoderm in the sea urchin gene regulatory network.

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

The control offoxN2/3expression in sea urchin embryos and its function in the skeletogenic gene regulatory network

Early development requires well-organized temporal and spatial regulation of transcription factors that are assembled into gene regulatory networks (GRNs). In the sea urchin, an endomesoderm GRN model explains much of the specification in the endoderm and mesoderm prior to gastrulation, yet some GRN connections remain incomplete. Here, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN state. Expression of foxN2/3 mRNA begins in micromeres at the hatched blastula stage and then is lost from micromeres at the mesenchyme blastula stage. foxN2/3 expression then shifts to the non-skeletogenic mesoderm and, later, to the endoderm. Here, we show that Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in micromeres. The later endomesoderm expression of foxN2/3 is independent of the earlier expression of foxN2/3 in micromeres and is independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of the larval skeleton. Early expression of genes for the skeletal matrix is dependent on FoxN2/3, but only until the mesenchyme blastula stage as foxN2/3 mRNA disappears from PMCs at that time and we assume that the protein is not abnormally long-lived. Knockdown of FoxN2/3 inhibits normal PMC ingression and foxN2/3 morphant PMCs do not organize in the blastocoel and fail to join the PMC syncytium. In addition, without FoxN2/3, the PMCs fail to repress the transfating of other mesodermal cells into the skeletogenic lineage. Thus, FoxN2/3 is necessary for normal ingression, for expression of several skeletal matrix genes, for preventing transfating and for fusion of the PMC syncytium.

Information processing at the foxa node of the sea urchin endomesoderm specification network

The foxa regulatory gene is of central importance for endoderm specification across Bilateria, and this gene lies at an essential node of the well-characterized sea urchin endomesoderm gene regulatory network (GRN). Here we experimentally dissect the cis-regulatory system that controls the complex pattern of foxa expression in these embryos. Four separate cis-regulatory modules (CRMs) cooperate to control foxa expression in different spatial domains of the endomesoderm, and at different times. A detailed mutational analysis revealed the inputs to each of these cis-regulatory modules. The complex and dynamic expression of foxa is regulated by a combination of repressors, a permissive switch, and multiple activators. A mathematical kinetic model was applied to study the dynamic response of foxa cis-regulatory modules to transient inputs. This study shed light on the mesoderm-endoderm fate decision and provides a functional explanation, in terms of the genomic regulatory code, for the spatial and temporal expression of a key developmental control gene.

Hedgehog signaling patterns mesoderm in the sea urchin

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.

Gene regulatory network interactions in sea urchin endomesoderm induction.

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.

Twist is an essential regulator of the skeletogenic gene regulatory network in the sea urchin embryo

Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.

Specification and patterning of the animal‐vegetal axis in sea urchins by the canonical Wnt signaling pathway

AbstractPattern formation along the sea urchin A‐V axis is initiated by the selective activation of the canonical Wnt signaling pathway in vegetal blastomeres. Activation of this pathway is essential for deployment of the endomesoderm gene regulatory network (EGRN), and for pattern formation along the entire A‐V axis. During early embryogenesis the canonical Wnt signaling pathway is selectively activated by Dishevelled (Dsh), a critical activator of the Wnt pathway. Dsh is highly enriched in vesicular structures at the vegetal pole in eggs and early embryos, and selective activation of this protein leads to the nuclearization of β‐catenin in the endomesoderm. Following activation of canonical Wnt signaling by Dsh, signaling by β‐catenin and the Lef/Tcf transcription factors regulates endomesoderm specification by activating the EGRN. One critical early target of nuclear β‐catenin is Wnt8, which is selectively expressed in the micromeres at the 16‐cell stage and in the macromeres one cleavage division later. Wnt8 signaling is not required for the endomesoderm‐inducing activity of the micromeres, but this protein regulates primary mesenchyme cell differentiation. Within the endomesodermal domain Wnt8 regulates the later specification of endoderm and mesoderm. These results have highlighted the important role of the canonical Wnt signaling pathway in patterning the A‐V axis in the sea urchin embryo, and have strongly suggested that this axis is initially specified by a cytoplasmic/cytoarchitectural mechanism to activate Dsh in vegetal blastomeres. Additionally, this work along with work in vertebrates and cnidarians has shown that the canonical Wnt pathway plays a conserved role in early pattern formation in metazoan embryos.

Deciphering the Underlying Mechanism of Specification and Differentiation: The Sea Urchin Gene Regulatory Network

The regulatory genome functions as a vast information processor through development. It processes the initial conditions that are set by asymmetric distributions of cellular components in the egg and translates them into the onset of spatially localized specification states. It regulates the timely differential activation of signaling molecules and transcription factors that divide the emerging domains into subdomains. It also governs the activation of groups of differentiation genes, the genes that encode, at the protein level, the functional and the structural properties of a cell type. The sea urchin endomesoderm gene regulatory network provides a window into the different levels of the regulatory apparatus. It demonstrates how the static physical genomic components define functional connections between the various regulatory genes that act together to conduct the dynamical developmental program.

Nemo-like kinase (NLK) acts downstream of Notch/Delta signalling to downregulate TCF during mesoderm induction in the sea urchin embryo

Studies in Caenorhabditis elegans and vertebrates have established that the MAP kinase-related protein NLK counteracts Wnt signalling by downregulating the transcription factor TCF. Here, we present evidence that during early development of the sea urchin embryo, NLK is expressed in the mesodermal precursors in response to Notch signalling and directs their fate by downregulating TCF. The expression pattern of nlk is strikingly similar to that of Delta and the two genes regulate the expression of each other. nlk overexpression, like ectopic activation of Notch signalling, provoked massive formation of mesoderm and associated epithelial mesenchymal transition. NLK function was found to be redundant with that of the MAP kinase ERK during mesoderm formation and to require the activity of the activating kinase TAK1. In addition, the sea urchin NLK, like its vertebrate counterpart, antagonizes the activity of the transcription factor TCF. Finally, activating the expression of a TCF-VP16 construct at blastula stages strongly inhibits endoderm and mesoderm formation, indicating that while TCF activity is required early for launching the endomesoderm gene regulatory network, it has to be downregulated at blastula stage in the mesodermal lineage. Taken together, our results indicate that the evolutionarily conserved TAK/NLK regulatory pathway has been recruited downstream of the Notch/Delta pathway in the sea urchin to switch off TCF-beta-catenin signalling in the mesodermal territory, allowing precursors of this germ layer to segregate from the endomesoderm.

Repression of mesodermal fate byfoxa, a key endoderm regulator of the sea urchin embryo

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Endomesoderm specification in Caenorhabditis elegans and other nematodes

The endomesoderm gene regulatory network (GRN) of C. elegans is a rich resource for studying the properties of cell-fate-specification pathways. This GRN contains both cell-autonomous and cell non-autonomous mechanisms, includes network motifs found in other GRNs, and ties maternal factors to terminal differentiation genes through a regulatory cascade. In most cases, upstream regulators and their direct downstream targets are known. With the availability of resources to study close and distant relatives of C. elegans, the molecular evolution of this network can now be examined. Within Caenorhabditis, components of the endomesoderm GRN are well conserved. A cursory examination of the preliminary genome sequences of two parasitic nematodes, Haemonchus contortus and Brugia malayi, suggests that evolution in this GRN is occurring most rapidly for the zygotic genes that specify blastomere identity.

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.

SoxB1 downregulation in vegetal lineages of sea urchin embryos is achieved by both transcriptional repression and selective protein turnover

Patterning of cell fates along the sea urchin animal-vegetal embryonic axis requires the opposing functions of nuclear beta-catenin/TCF-Lef, which activates the endomesoderm gene regulatory network, and SoxB1, which antagonizes beta-catenin and limits its range of function. A crucial aspect of this interaction is the temporally controlled downregulation of SoxB1, first in micromeres and then in macromere progeny. We show that SoxB1 is regulated at the level of protein turnover in these lineages. This mechanism is dependent on nuclear beta-catenin function. It can be activated by Pmar1, but not by Krl, both of which function downstream of beta-catenin/TCF-Lef. At least partially distinct, lineage-specific mechanisms operate, as turnover in the macromeres depends on entry of SoxB1 into nuclei, and on redundant destruction signals, neither of which is required in micromeres. Neither of these turnover mechanisms operates in mesomere progeny, which give rise to ectoderm. However, in mesomeres, SoxB1 appears to be subject to negative autoregulation that helps to maintain tight regulation of SoxB1 mRNA levels in presumptive ectoderm. Between the seventh and tenth cleavage stages, beta-catenin not only promotes degradation of SoxB1, but also suppresses accumulation of its message in macromere-derived blastomeres. Collectively, these different mechanisms work to regulate precisely the levels of SoxB1 in the progeny of different tiers of blastomeres arrayed along the animal-vegetal axis.

LvGroucho and nuclear β-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo

In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize β-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and nβ-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with β-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.

An otx cis-regulatory module: a key node in the sea urchin endomesoderm gene regulatory network

An essential node in the gene regulatory network (GRN) for endomesoderm specification in the sea urchin embryo lies within the regulatory system of the otx gene. According to the predictions of the GRN, based on perturbation analysis and expression data, the β1/2 transcription unit of this gene is engaged during specification in interactions with two other regulatory genes, krox and gatae. It is predicted to be driven into activity by the krox gene, to form a positively reinforcing functional loop with the gatae gene, and to respond to its own output as well. Here we isolate the relevant otx cis-regulatory element, and examine the specific input predictions of the GRN at the level of its genomic DNA sequence. This β1/2-otx regulatory module performs the necessary functions, as shown by use of expression constructs. It requires gatae, otx, and krox inputs, as predicted, and it operates as an “AND” logic processor in that removal of any one of these inputs essentially destroys activity. The necessary target sites were identified in the module sequence, and mutation of these sites was demonstrated to produce the same respective effects on construct expression as does blocking its regulatory inputs by treatment with morpholino antisense oligonucleotides. For spatial expression in the endoderm, one particular pair of Gata sites is essential and these function synergistically with an adjacent Otx site. We thus demonstrate directly the structure/function relationships of the genomic regulatory code, at this key node of the endomesoderm GRN.