Glyconectin Cell Adhesion Epitope, ß-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis.

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge that connects the two newly formed sibling cells, which is ultimately resolved by abscission. While mitosis is completed within 15 min, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell-cell communication is fundamental for development, the role of intercellular bridges during embryogenesis has not been fully elucidated. In this work, we characterized the spatiotemporal characteristics of the intercellular bridge during early zebrafish development. We found that abscission is delayed during the rapid division cycles that occur in the early embryo, giving rise to the formation of interconnected cell clusters. Abscission was accelerated when the embryo entered the midblastula transition (MBT) phase. Components of the ESCRT machinery, which drives abscission, were enriched at intercellular bridges post-MBT and, interfering with ESCRT function, extended abscission beyond MBT. Hallmark features of MBT, including transcription onset and cell shape modulations, were more similar in interconnected sibling cells compared to other neighboring cells. Collectively, our findings suggest that delayed abscission in the early embryo allows clusters of cells to coordinate their behavior during embryonic development.

Genetic developmental timing revealed by inter-species transplantations in fish.

The path from a fertilised egg to an embryo involves the coordinated formation of cell types, tissues and organs. Developmental modules comprise discrete units specified by self-sufficient genetic programs that can interact with each other during embryogenesis. Here, we have taken advantage of the different span of embryonic development between two distantly related teleosts, zebrafish (Danio rerio) and medaka (Oryzias latipes) (3 and 9 days, respectively), to explore modularity principles. We report that inter-species blastula transplantations result in the ectopic formation of a retina formed by donor cells - a module. We show that the time taken for the retina to develop follows a genetic program: an ectopic zebrafish retina in medaka develops with zebrafish dynamics. Heterologous transplantation results in a temporal decoupling between the donor retina and host organism, illustrated by two paradigms that require retina-host interactions: lens recruitment and retino-tectal projections. Our results uncover a new experimental system for addressing temporal decoupling along embryonic development, and highlight the presence of largely autonomous but interconnected developmental modules that orchestrate organogenesis.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development.

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.

Embryonic and early larval development of two marine angelfish, Centropyge bicolor and Centropyge bispinosa.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 µm for C. bicolor and 627.6 ± 7.8 µm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

Evaluation of BMP-mediated patterning in a 3D mathematical model of the zebrafish blastula embryo.

Bone Morphogenetic Proteins (BMPs) play an important role in dorsal-ventral (DV) patterning of the early zebrafish embryo. BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however it is not clear how the source-sink mechanism shapes patterns in 3D, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo. We propose a new three-dimensional growing Partial Differential Equation (PDE)-based model to simulate the BMP patterning process during the blastula stage. This model provides a starting point to elucidate how different mechanisms and components work together in 3D to create and maintain the BMP gradient in the embryo. We also show how the 3D model fits the BMP signaling gradient data at multiple time points along both axes. Furthermore, sensitivity analysis of the model suggests that the spatiotemporal patterns of Chordin and BMP ligand gene expression are dominant drivers of shape in 3D and more work is needed to quantify the spatiotemporal profiles of gene and protein expression to further refine the models.

Altering the levels of nuclear import factors in early Xenopus laevis embryos affects later development.

More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.

Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis.

During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.

Differentially-dimensioned furrow formation by zygotic gene expression and the MBT.

Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germ-band types.

BACKGROUND: Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. RESULTS: We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. CONCLUSION: miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

Both Nuclear Size and DNA Amount Contribute to Midblastula Transition Timing in Xenopus laevis.

During early Xenopus laevis embryogenesis both nuclear and cell volumes decrease with the nuclear-to-cytoplasmic (N/C) volume ratio reaching a maximum at the midblastula transition (MBT). At the MBT, embryonic transcription is upregulated and cell cycles lengthen. Early studies demonstrated a role for the DNA-to-cytoplasmic ratio in the control of MBT timing. By altering nuclear size, we previously showed that the N/C volume ratio also contributes to proper MBT timing. Here we examine the relative contributions of nuclear size and DNA amount to MBT timing by simultaneously altering nuclear size and ploidy in X. laevis embryos. Compared to diploid embryos, haploids exhibited a delay in both zygotic gene expression and cell cycle lengthening, while diploid embryos with increased N/C volume ratios showed early expression of zygotic genes and premature lengthening of cell cycles. Interestingly, haploids with increased N/C volume ratios exhibited an intermediate effect on the timing of zygotic gene expression and cell cycle lengthening. Decreasing nuclear size in post-MBT haploid embryos caused a further delay in cell cycle lengthening and the expression of some zygotic genes. Our data suggest that both the N/C volume ratio and DNA amount contribute to the regulation of MBT timing with neither parameter being dominant.

Origin of the vertebrate body plan via mechanically biased conservation of regular geometrical patterns in the structure of the blastula.

We present a plausible account of the origin of the archetypal vertebrate bauplan. We offer a theoretical reconstruction of the geometrically regular structure of the blastula resulting from the sequential subdivision of the egg, followed by mechanical deformations of the blastula in subsequent stages of gastrulation. We suggest that the formation of the vertebrate bauplan during development, as well as fixation of its variants over the course of evolution, have been constrained and guided by global mechanical biases. Arguably, the role of such biases in directing morphology-though all but neglected in previous accounts of both development and macroevolution-is critical to any substantive explanation for the origin of the archetypal vertebrate bauplan. We surmise that the blastula inherently preserves the underlying geometry of the cuboidal array of eight cells produced by the first three cleavages that ultimately define the medial-lateral, dorsal-ventral, and anterior-posterior axes of the future body plan. Through graphical depictions, we demonstrate the formation of principal structures of the vertebrate body via mechanical deformation of predictable geometrical patterns during gastrulation. The descriptive rigor of our model is supported through comparisons with previous characterizations of the embryonic and adult vertebrate bauplane. Though speculative, the model addresses the poignant absence in the literature of any plausible account of the origin of vertebrate morphology. A robust solution to the problem of morphogenesis-currently an elusive goal-will only emerge from consideration of both top-down (e.g., the mechanical constraints and geometric properties considered here) and bottom-up (e.g., molecular and mechano-chemical) influences.

A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology.

[Surface Microdeformations and Regulation of Cell Movements in Xenopus Development].

The velocities and directions of movements of individual outer ectodermal cells of Xenopus embryos in the course of normal development from the blastula to the early tail-bud stage, as well as after mechanical relaxation in the early gastrula, were measured. An alternation of the periods of directed movements of large cell masses and local cell wanderings was detected. In both cases, the trajectories of individual cells consisted primarily of orthogonal segments. Cell movements were measured on two scales. At a small-scale consideration (time intervals of the order of several hours and distances of the order of tens of microns), fairly slight linear stretching and compressive deformations were detected, which looked like gentle smooth gradients along which the upward morphogenetic movements of cells were directed. At a large-scale consideration (time intervals of the order of tens of minutes and distances of the order of microns), quasi-periodic fluctuations of velocities of individual cells partly correlated in time were found. The differences between these velocities generated microdeformations, which reached several tens of percent and developed within time intervals not more than 10 min. Measurements of relative magnitudes of mechanical forces influencing the cell walls suggests that microdeformations generate local stretching and compressive deformations modulating smoother tension gradients.

Roles of hesC and gcm in echinoid larval mesenchyme cell development.

To understand the roles of hesC and gcm during larval mesenchyme specification and differentiation in echinoids, we performed perturbation experiments for these genes in two distantly related euechinoids, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. The number of larval mesenchyme cells increased when the translation of hesC was inhibited, thereby suggesting that hesC has a general role in larval mesenchyme development. We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. Simultaneous inhibition of the translation of hesC and gcm induced a significant increase in the number of skeletogenic cells, which suggests that gcm functions in skeletogenic fate repression. Based on these observations, we suggest that: (i) hesC participates in some general aspects of mesenchymal cell development; and (ii) gcm is involved in the mechanism responsible for the binary specification of skeletogenic and pigment cell fates.

An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo.

Anterior signaling centers help specify and pattern the early anterior neuroectoderm (ANE) in many deuterostomes. In sea urchin the ANE is restricted to the anterior of the late blastula stage embryo, where it forms a simple neural territory comprising several types of neurons as well as the apical tuft. Here, we show that during early development, the sea urchin ANE territory separates into inner and outer regulatory domains that express the cardinal ANE transcriptional regulators FoxQ2 and Six3, respectively. FoxQ2 drives this patterning process, which is required to eliminate six3 expression from the inner domain and activate the expression of Dkk3 and sFRP1/5, two secreted Wnt modulators. Dkk3 and low expression levels of sFRP1/5 act additively to potentiate the Wnt/JNK signaling pathway governing the positioning of the ANE territory around the anterior pole, whereas high expression levels of sFRP1/5 antagonize Wnt/JNK signaling. sFRP1/5 and Dkk3 levels are rigidly maintained via autorepressive and cross-repressive interactions with Wnt signaling components and additional ANE transcription factors. Together, these data support a model in which FoxQ2 initiates an anterior patterning center that implements correct size and positions of ANE structures. Comparisons of functional and expression studies in sea urchin, hemichordate and chordate embryos reveal striking similarities among deuterostome ANE regulatory networks and the molecular mechanism that positions and defines ANE borders. These data strongly support the idea that the sea urchin embryo uses an ancient anterior patterning system that was present in the common ambulacrarian/chordate ancestor.