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The diadematid sea urchin Centrostephanus rodgersii occurs in Australia and New Zealand and has undergone recent southward range extension in Australia as a result of regional warming. Clarifying the population genetic structure of this species across its New Zealand range would allow a better understanding of recent and future mechanisms driving range changes in the species. Here, we use microsatellite DNA data to assess connectivity and genetic structure in 385 individuals from 14 locations across the Australian and New Zealand ranges of the species. We detected substantial genetic differentiation among C. rodgersii populations from Australia and New Zealand. However, the population from Port Stephens (located north of Newcastle), Australia, strongly clustered with New Zealand samples. This suggests that the New Zealand populations recently originated from this area, likely via larval transport in the Tasman Front flow that arises in this region. The weak population genetic structure and relatively low genetic diversity detected in New Zealand (global F.sub.st = 0.0021) relative to Australia (global F.sub.st = 0.0339) is consistent with the former population's inferred history of recent climate-driven expansion. Population-level inbreeding is low in most populations, but were higher in New Zealand (global F.sub.is = 0.0833) than in Australia (global F.sub.is = 0.0202), suggesting that self-recruitment is playing an increasingly important role in the New Zealand region. Our results suggest that C. rodgersii is likely to spread southwards as ocean temperatures increase; therefore, it is crucial that researchers develop a clearer understanding of how New Zealand ecosystems will be reshaped by this species (and others) under climate change., Author(s): L. J. Thomas [sup.1] , L. Liggins [sup.2] [sup.3] , S. C. Banks [sup.4] , L. B. Beheregaray [sup.5] , M. Liddy [sup.1] , G. A. McCulloch [sup.6] , [...]

Abstract: Accumulating evidence indicates that Kruppel-like factors (KLFs) play important roles in fat biology via the regulation of CCAAT/enhancer binding proteins (C/EBPs). However, KLFs and C/EBPs have not been identified [...]

Abstract. Planktotrophic sea urchin larvae are developmentally plastic: in response to food scarcity, development of the juvenile rudiment is suspended and larvae instead develop elongated arms, thus increasing feeding capacity [...]

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. cis-regulatory analysis | gene regulatory networks | mathematical modeling | embryonic development doi/ 10.1073/pnas.1004824107

Gene expression is controlled by interactions between trans-regulatory factors and cis-regulatory DNA sequences, and these interactions constitute the essential functional linkages of gene regulatory networks (GRNs). Validation of GRN models requires experimental cis-regulatory tests of predicted linkages to authenticate their identities and proposed functions. However, cis-regulatory analysis is, at present, at a severe bottleneck in genomic system biology because of the demanding experimental methodologies currently in use for discovering cis-regulatory modules (CRMs), in the genome, and for measuring their activities. Here we demonstrate a high-throughput approach to both discovery and quantitative characterization of CRMs. The unique aspect is use of DNA sequence tags to 'barcode' CRM expression constructs, which can then be mixed, injected together into sea urchin eggs, and subsequently deconvolved. This method has increased the rate of cis-regulatory analysis by > 100-fold compared with conventional one-by-one reporter assays. The utility of the DNA-tag reporters was demonstrated by the rapid discovery of 81 active CRMs from 37 previously unexplored sea urchin genes. We then obtained simultaneous high-resolution temporal characterization of the regulatory activities of more than 80 CRMs. On average 2-3 CRMs were discovered per gene. Comparison of endogenous gene expression profiles with those of the CRMs recovered from each gene showed that, for most cases, at least one CRM is active in each phase of endogenous expression, suggesting that CRM recovery was comprehensive. This approach will qualitatively alter the practice of GRN construction as well as validation, and will impact many additional areas of regulatory system biology. high-throughput discovery | sea urchin gene regulation doi/10.1073/pnas.1000147107

Design features that ensure reproducible and invariant embryonic processes are major characteristics of current gene regulatory network models. New cis-regulatory studies on a gene regulatory network subcircuit activated early in the development of the sea urchin embryo reveal a sequence of encoded 'fail-safe' regulatory devices. These ensure the maintenance of fate separation between skeletogenic and nonskeletogenic mesoderm lineages. An unexpected consequence of the network design revealed in the course of these experiments is that it enables the embryo to 'recover' from regulatory interference that has catastrophic effects if this feature is disarmed. A reengineered regulatory system inserted into the embryo was used to prove how this system operates in vivo. Genomically encoded backup control circuitry thus provides the mechanism underlying a specific example of the regulative development for which the sea urchin embryo has long been famous. gene regulatory | embryonic development | regulatory subcircuit topology | pmarlhesC doi/10.1073/pnas.0910007106

Strongylocentrotus intermedius (A. Agassiz, 1863) is an economically important sea urchin inhabiting the northwest Pacific region of Asia. The northern Primorye (Sea of Japan) populations of S. intermedius consist of two sympatric morphological forms, 'usual' (U) and 'gray' (G). The two forms are significantly different in morphology and preferred bathymetric distribution, the G form prevailing in deeper-water settlements. We have analyzed the genetic composition of the S. intermedius forms using the nucleotide sequences of the mitochondrial gene encoding the cytochrome c oxidase subunit I and the nuclear gene encoding bindin to evaluate the possibility of cryptic species within S. intermedius. We have examined the presence of symbiont microorganisms by means of 16S rRNA sequences. The nucleotide sequence divergence between the morphological forms is Iow: 0.74% and 0.70% for cytochrome c oxidase subunit I and nuclear gene encoding bindin, respectively, which is significantly below average intrageneric sequence divergence among Strongylocentrotus species. We thus have found no genetic evidence of cryptic species within S. intermedius. Phylogenetic analysis shows that the bacteria symbionts of S. intermedius belong to the phylum Bacteroidetes, but the U and G forms predominantly harbor highly divergent bacterial lineages belonging to two different taxonomic classes, Flavobacteria and Sphingobacteria. We propose that the U and G forms of S. intermedius represent distinct ecomorphological adaptations to contrasting shallow- and deep-water marine environments and might be considered incipient species. We also propose that the symbiotic bacteria likely play an important role in the evolution of morphological divergence of S. intermedius. Bacteroidetes | DNA polymorphism | incipient speciation | marine adaptation | sympatric morphological forms

Of the five echinoderm classes, only the modern sea urchins (euechinoids) generate a precociously specified embryonic micromere lineage that ingresses before gastrulation and then secretes the biomineral embryonic skeleton. The gene regulatory network (GRN) underlying the specification and differentiation of this lineage is now known. Many of the same differentiation genes as are used in the biomineralization of the embryo skeleton are also used to make the similar biomineral of the spines and test plates of the adult body. Here, we determine the components of the regulatory state upstream of these differentiation genes that are shared between embryonic and adult skeletogenesis. An abrupt 'break point' in the micromere GRN is thus revealed, on one side of which most of the regulatory genes are used in both, and on the other side of which the regulatory apparatus is entirely micromere specific. This reveals the specific linkages of the micromere GRN forged in the evolutionary process by which the skeletogenic gene batteries were caused to be activated in the embryonic micromere lineage. We also show, by comparison with adult skeletogenesis in the sea star, a distant echinoderm outgroup, that the regulatory apparatus responsible for driving the skeletogenic differentiation gene batteries is an ancient pleisiomorphic aspect of the echinoderm-specific regulatory heritage. embryonic and adult skeletogenesis | gene network evolution | sea urchin | sea star

Explanation of a process of development must ultimately be couched in the terms of the genomic regulatory code. Specification of an embryonic cell lineage is driven by a network of interactions among genes encoding transcription factors. Here, we present the gene regulatory network (GRN) that directs the specification of the skeletogenic micromere lineage of the sea urchin embryo. The GRN now includes all regulatory genes expressed in this lineage up to late blastula stage, as identified in a genomewide survey. The architecture of the GRN was established by a large-scale perturbation analysis in which the expression of each gene in the GRN was cut off by use of morpholinos, and the effects on all other genes were measured quantitatively. Several cis-regulatory analyses provided additional evidence. The explanatory power of the GRN suffices to provide a causal explanation for all observable developmental functions of the micromere lineage during the specification period. These functions are: (i) initial acquisition of identity through transcriptional interpretation of localized maternal cues; (ii) activation of specific regulatory genes by use of a double negative gate; (iii) dynamic stabilization of the regulatory state by activation of a feedback subcircuit; (iv) exclusion of alternative regulatory states; (v) presentation of a signal required by the micromeres themselves and of two different signals required for development of adjacent endomesodermal lineages; and (vi) lineage-specific activation of batteries of skeletogenic genes. The GRN precisely predicts gene expression responses and provides a coherent explanation of the biology of specification. gene regulatory networks | network subcircuits | sea urchin embryo | skeletogenic micromeres

Understanding the scale of marine population connectivity is critical for the conservation and sustainable management of marine resources. For many marine species adults are benthic and relatively immobile, so patterns of larval dispersal and recruitment provide the key to understanding marine population connectivity. Contrary to previous expectations, recent studies have often detected unexpectedly low dispersal and fine-scale population structure in the sea, leading to a paradigm shift in how marine systems are viewed. Nonetheless, the link between fine-scale marine population structure and the underlying physical and biological processes has not been made. Here we show that patterns of genetic structure and population connectivity in the broadcast-spawning and long-distance dispersing sea urchin Centrostephanus rodgersii are influenced by physical oceanographic and geographic variables. Despite weak genetic differentiation and no isolation-by-distance over thousands of kilometers among samples from eastern Australia and northern New Zealand, fine-scale genetic structure was associated with sea surface temperature (SST) variability and geography along the southeastern Australian coast. The zone of high SST variability is characterized by periodic shedding of eddies from the East Australian Current, and we suggest that ocean current circulation may, through its influence on larval transport and recruitment, interact with the genetic consequences of large variance in individual reproductive success to generate patterns of fine-scale patchy genetic structure. If proven consistent across species, our findings suggest that the optimal scale for fisheries management and reserve design should vary among localities in relation to regional oceanographic variability and coastal geography. Key words: Centrostephanus rodgersii; connectivity; genetic patchiness; larval dispersal; microsatellite; multilocus spatial autocorrelation; oceanography; sea urchin.

Abstract: In this paper the chromosomal localization and molecular cloning and characterization of three 5S rDNA clusters of 700 bp (base pairs), 900 bp, and 950 bp in the sea [...]

A study examines the genome sequencing of sea urchins by using microarray analysis.

A study examines the protein domains in the genome of sea urchin. The genes of sea urchin is compared with the genes of other vertebrates.

A study examines opsin proteins found in the genome of sea urchin. The genomic clustering and gene structure of the urchin is discussed.

A study on neural genes examines the genome sequencing of strongylocentrotus purpuratus.

A study on the sea urchin egg examines the role of calcium in egg fertilization. Genome sequencing is done to study fertilization.

A study examines the germ line determination of sea urchin strongylocentrotus purpuratus.

A study examines the oocyte functions that are responsible for cell development in sea urchin.

A study examines the genes of purple sea urchins responsible for its immunity.

A study on the death of cell of sea urchins examines the proteins found in its genome.

A study examines the metazoan genomes of sea urchin strongylocentrotus purpuratus which responds to environmental stressors.

A study on cellular mechanism of sea urchin examines its biomineralization process.

A study examines the histone genes found in the embryo and larvae of the sea urchin.

A study on the embryonic development of sea urchin examines the in-silico screening of the genome.

A study on the enzymatic development of sea urchin strongylocentrotus purpuratus examines its genome sequencing.

A study makes a microarray analysis to examine the genes of sea urchin strongylocentrotus purpuratus. The matrix metalloproteases and disintegrin metalloproteases families are examined.

A study examines the cellular development in the embryo of sea urchin. Cell adhesion and migration are discussed.

A study on the genome of sea urchins strongylocentrotus purpuratus examines its cellular development and DNA metabolism.

A study examines the role of cytoskeleton in the embryonic development of sea urchin. Several gene families of the sea urchin are compared.

A study examines the protein kinases seen in the sea urchin kinome and compares them with the kinases found in human beings.

A study examines protein phosphatases found in the genome of sea urchin Strongylocentrotus purpuratus. The role of phosphatases in signaling pathways is discussed.

A study on the cellular development and genome sequencing of sea urchins examines their hedgehog and notch pathways. The genetic expression of the sea urchin is studied.

A study examines the fox genes found in the genome of sea urchin as well as its embryonic development. The transcription factors of the fox family is analyzed.

A study on the development of sea urchin embryo examines the transcription factor usage. Regulation of genes and its bilaterian development are discussed.

Many evolutionary modifications in development and life history derive from changes in embryonic gene expression. However, the genetic variation affecting gene expression in natural populations is not well understood, nor are the evolutionary mechanisms that operate on that variation. The early embryonic gene network of the purple sea urchin (Strongylocentrotus purpuratus) has been studied in considerable detail, providing an informative basis for analyzing the developmental and evolutionary mechanisms that alter gene expression. Comparative functional analyses have been carried out for several genes. These case studies indicate a complex relationship between sequence divergence and gene expression: in some cases, gene expression is conserved despite extensive divergence in cis-regulatory sequences, while in others the basis for a change in gene expression does not reside locally but rather in the expression or activity of transcription factors that regulate its expression. Diverse evolutionary mechanisms apparently operate on cis-regulatory regions, including negative, balancing, and stabilizing selection.

Fatty acid analyses are emerging as a powerful technique to probe trophic interactions between organisms. In this paper, the application of both this procedure and gonad index (GI) determination on two populations (intertidal and subtidal) of the echinoid Psammechinus miliaris is reported. The investigation spanned the 3-month spawning period of Scottish west coast populations. In both populations a progressive decrease in the GI was found, coupled with an increasing maturity stage (from mature to spent). Sexual maturation and decrease in GI was synchronous between the two populations. In conjunction, there were distinct changes in gonad biochemistry. Differences in the fatty acid composition of the gonad reflected the changes in sexual maturation. Mature males and females had significant differences in the fatty acid composition of their gonads, whereas post-spawned individuals showed no gender differences. Male urchins had higher levels of polyunsaturated fatty acids (PUFAs) compared to females, and there was a dramatic reduction in the fatty acids 22:6(n-3) and 20:5(n-3) with increasing maturity stage. Using multivariate statistical techniques, these changes in the fatty acid composition of the sea urchin gonad were linked to habitat related diet differences combined with gender differences. These changes in the fatty acid signatures clearly reflect the dual function of the gonad as both a nutrient store and a reproductive organ., Introduction Understanding the trophic relationships that exist within and between species, and the way that these interactions vary along temporal and spatial scales has long been a principle component of [...]

Strongylocentrotus purpuratus is used to demonstrate the wnt8 gene's modular cis-regulatory system and its impact on normal endomesodermal expression and expression in macromere and micromere descendant cells in early cleavage.

The role of sea urchin dual oxidase Udx1 in blocking polyspermy is described. Cleavage is delayed if this enzyme is inhibited. Udx1 may also have a role in intracellular signaling.

We demonstrated that environmentally relevant levels of UVA and UVB can reduce sperm motility (UVA: by 38-58%; UVB: by 42 85%; P < 0.05) and subsequently fertilisation success (UVA: by 38-72%; UVB: by 91-98%; P < 0.05) of urchins (Anthocidaris crassispina) in a dose-dependent manner, implicating that recruitment of urchin populations might be reduced by UVR (ultraviolet radiation) prevailing in their natural habitats. Concomitantly, reactive oxygen species (ROS) production was enhanced by UVA and UVB in a dose-dependent manner (UVA: by 1.3-fold; UVB: by 6.6-7.3-fold; P < 0.05), and the increase in ROS resulted in an increase in lipid peroxidation (LPO) in urchin sperm (UVA: by 4.2-7.2-fold: UVB: by 2.3-2.7fold; P < 0.05). This study demonstrated that ROS production and oxidative damages enhanced by UVR may account for the observed declines in sperm motility and fertilisation, and suggests that levels of UVR prevailing in the environment may pose a significant threat to the reproductive success of natural populations of urchins spawning in shallow waters.

During Strongylocentrotus purpuratus embryogenesis, aboral ectoderm-specific expression of spec2a relies on an upstream enhancer that confers its spatial specificity largely through repression. The purpose of this study was to determine how spec2a expression is repressed in endoderm and oral ectoderm territories. A 78-base pair DNA sequence within the enhancer contains five tightly spaced cis-regulatory elements including proximal (TAATCT) and distal (TAATCC) elements that bind to both SpOtx, a broadly distributed transcriptional activator, and SpGoosecoid (SpGsc), an oral ectoderm-restricted transcriptional repressor. We show here that these two seemingly redundant Otx/Gsc elements have distinct functions. The proximal element bound to SpGATA-E, an endomesoderm-specific transcription factor. Treatment with SpGATA-E and SpGsc morpholino antisense oligonucleotides (MASOs) resulted in enhanced transcriptional activity from the proximal element, suggesting that both factors functioned as repressors at this site. SpGATA-E MASO-treated embryos failed to express ectoderm markers, indicating a role for SpGATA-E in ectoderm differentiation. The spec2a proximal element was distinct from the corresponding element in the related spec1 enhancer, and swaps between spec1 and spec2a cis-regulatory elements indicated, that for optimal repression, the proximal element had to interact with a nearby CCAAT-binding factor element. Our results show that the recently evolved proximal element contributes to the repression of spec2a in endomesoderm and oral ectoderm territories. Keywords: Sea urchin development; Transcriptional repression; cis-Regulatory elements; GATA; Goosecoid; Otx

The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. Here, we identify critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes. We found multiple copies of a repetitive sequence element termed RSR in genomes of species within the Strongylocentrotidae family, but RSRs were not detected in genomes of species outside Strongylocentrotidae. spec genes in Strongylocentrotus purpuratus are invariably associated with RSRs, and the spec2a RSR functioned as a transcriptional enhancer and displayed greater activity than did spec1 or spec2c RSRs. Single-base pair differences at two cis-regulatory elements within the spec2a RSR increased the binding affinities of four transcription factors, SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which these four factors bound were recent evolutionary acquisitions that acted to either activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. Our results indicated that a dynamic pattern of cis-regulatory element evolution exists for spec genes despite their conserved aboral ectoderm expression. Keywords: cis-regulatory element evolution; Stabilizing selection; Sea urchin evolution; Sea urchin development; Sea urchin genomics; spec gene; Otx; Goosecoid; CBF; GATA

Sea urchin eggs secrete a serine protease activity, CGSPI, at fertilization that is essential for the block to polyspermy. Several targets of this proteolytic activity on the plasma membrane were identified here using a cell surface biotinylation approach. Amino acid microsequencing of one of these proteins led to the identification of a 4.75-kb cDNA clone from a Strongylocentrotus purpuratus ovary eDNA library that encodes a 160-kDa protein called p160. This protein contains five CUB domains and a putative transmembrane domain suggesting that p160 is an integral membrane protein with protein protein interaction motifs facing the extracellular matrix of the egg. Whole-mount immunolocalization studies demonstrate that p160 is on the surface of the egg, enriched at the tips of microvilli. The protein is removed at fertilization in a protease-dependent manner, and functional assays suggest that p160 serves to link the plasma membrane to the vitelline layer until fertilization. Thus, p 160 is a key candidate for a vitelline-layer linker protein, the selective proteolysis of which functions in the block to polyspermy in the sea urchin egg. Keywords: Block to polyspermy; Egg; Fertilization; Protease; Sea urchin; Strongylocentrotus purpuratus: Vitelline layer; CGSP1

Mesoderm and mesodermal structures in the sea urchin embryo are entirely generated by two embryologically distinct populations of mesenchyme cells: the primary (PMC) and the secondary (SMC) mesenchyme cells. We have identified the extracellular signal-regulated kinase (ERK) as a key component of the regulatory machinery that controls the formation of both these cell types. ERK is activated in a spatial-temporal manner, which coincides with the epithelial--mesenchyme transition (EMT) of the prospective PMCs and SMCs. Here, we show that ERK controls EMT of both primary and secondary mesenchyme cells. Loss and gain of function experiments demonstrate that ERK signaling is not required for the early specification of either PMCs or SMCs, but controls the maintenance and/or the enhancement of expression levels of regulatory genes which participate in the process of specification of these cell types. In addition, ERK-mediated signaling is essential for the transcription of terminal differentiation genes encoding proteins that define the final structures generated by PMCs and SMCs. Our findings suggest that ERK has a central pan-mesodermal role in coupling EMT and terminal differentiation of all mesenchymal cell types in the sea urchin embryo. Keywords: MAP kinase; ERK; MEK; ets; erg; Epithelial-mesenchyme transition; Sea urchin embryo

Various hybrids of sea urchin reveal their pace of evolution and development. It is observed that hybridizations among direct and indirect developing sea urchins differing in developmental phenotype in parallel with investigations of genetic programs will identify the quantity of pluteal development lost before it is rescued in such crosses.

In Japan, mass-production of sea urchin juveniles involves the culture of periphytic diatom films on plastic plates in 5- to 15-tonne tanks for the induction of larval metamorphosis. This study focused on the larval response of sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina, to natural microbial films in the sea and diatom-based films formed in the tanks. The effect of diatoms and bacteria on larval metamorphosis was also examined using laboratory-cultured diatom-based films in the presence of germanium dioxide and antibiotics during film culture. Furthermore, the nature of the cue of the cultured diatom-based film was also investigated. Results showed that P. depressus and A. crassispina metamorphosed both on natural microbial films and diatom-based films in a tank. In the sea, the metamorphosis (%) of P. depressus increased gradually in accordance with the immersion period of film formed on glass slides, whereas the larval metamorphosis of A. crassispina had a bell-shaped response curve. In the tank, although the diatom-based films showed a low inducing activity for larval metamorphosis of A. crassispina, the metamorphosis of P. depressus larvae increased linearly in accordance with the diatom density. These results suggest that diatom-based films could promote the larval metamorphosis of P. depressus, but are less important in A. crassispina. In a simultaneous larval assay (May), P. depressus showed a higher percentage of metamorphosis than A. crassispina. We concluded that the former is more sensitive to diatombased film than the latter and that this is due to differences in their natural habitats. For laboratory-cultured diatom-based film, both species of sea urchins showed a similar response, in which reduction in diatom and bacteria density resulted in a decrease in the original inducing activity. There seems to be a synergistic effect between diatom and bacteria in inducing larval metamorphosis. Films subjected to treatment with 0.1 N HCl were no longer inductive for either sea urchin, while those films treated with 40[degrees]C heat or EtOH (5% and 10% EtOH) showed a significant reduction in the inducing activity. Thus the surface-associated cues may be highly susceptible to the above treatments., Introduction Most marine invertebrates have a planktonic larval stage preceding a benthic adult phase. The selection of a suitable habitat by the settling larvae often determines the long-term survival of [...]

In the sea urchin embryo skeletogenesis is the result of a complex series of molecular and cellular events that coordinate the morphogenetic process. Past and recent evidence strongly indicate that skeletal initiation and growth are strictly dependent on signals emanating from the oral ectodermal wall. As previously suggested, Orthopedia (Otp), a homeodomain-containing transcription factor specifically expressed in a small subset of oral ectoderm cells, might be implicated in this signaling pathway. In this study, we utilize three different strategies to address the issue of whether Otp is an upstream regulator of sketelogenesis. We describe the effects of microinjection of Otp morpholino-substituted antisense oligonucleotides and dominant-negative Otp-engrailed mRNA in Paracentrotus lividus embryos. We demonstrate that inhibition of Otp expression completely abolishes skeletal synthesis. By contrast, coinjection of Otp mRNA and the morpholino antisense oligonucleotide specifically rescues the skeletogenic program. In addition, localized ectodermal expression of the Otp-GFP fusion gene construct driven by the hatching enzyme promoter, induces ectopic and abnormal spiculogenesis. We further show that an indirect target of this homeoprotein is the skeletogenic specific gene SM30, whose expression is known to be under the strict control of the oral ectoderm territory. Based on these results, we conclude that Otp triggers the ectoderm-specific signal that promotes skeletogenesis. Keywords: Orthopedia homeobox; Sea urchin embryo; Skeletogenesis; Morpholino oligonucleotides

Previous studies in sea urchin embryos have demonstrated that nuclearization of [beta]-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by [beta]-catenin-dependent gene products that include the transcription repressor SpKr1. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of [beta]-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-[beta]-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow [beta]-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation. Keywords: Morpholino; Misexpression; Animal-vegetal axis

The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an [alpha]Spdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with [alpha]Spdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of [alpha]Spdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained [alpha]Spdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in [alpha]Spdri MASO embryos. Part of its cause is interference by [alpha]Spdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect. Keywords: Gene regulatory network; Oral ectoderm; deadringer; Sea urchin embryo

pmarl is a transcription factor in the paired class homeodomain family that was identified and found to be transcribed in micromeres beginning at the fourth cleavage of sea urchin development [Dev. Biol. 246 (2002), 209]. Based on in situ data, molecular perturbation studies, and QPCR data, the recently published gene regulatory network (GRN) model for endomesoderm specification [Science 295 (2002) 1669; Dev. Biol. 246 (2002), 162] places pmarl early in the micromere specification pathway, and upstream of two important micromere induction signals. The goal of this study was to test these three predictions of the network model. A series of embryo chimeras were produced in which pmarl activity was perturbed in one cell that was transplanted to control hosts. At the fourth cleavage, micromeres bearing altered pmarl activity were combined with a normal micromereless host embryo. If [beta]-catenin signaling is blocked, the micromeres remain unspecified and are unable to signal to the host cells. When such [beta]-catenin-blocked micromeres also express Pmarl, all observed micromere functions are rescued. The rescue includes expression of the primary mesenchyme cell (PMC) differentiation program, expression and execution of the Delta signal to induce secondary mesoderm cell (SMC) specification in macromere progeny, and expression of the early endomesoderm induction signal necessary for full specification of the endoderm. Additionally, Pmarl expressed mosaically from inserted DNA constructs causes induction of ectopic Endo 16 in adjacent cells, demonstrating further that Pmarl controls expression of the early endomesoderm induction signal. Based on these experiments, Pmarl is an important transcription factor necessary for initiating the micromere specification program and for the expression of two inductive signals produced by micromeres. Each of the tests we describe supports the placement and function of Pmarl in the endomesoderm GRN model. Keywords: Gene regulatory network; Sox; Delta; [beta]-Catenin; Paired homeodomain

Research has been conducted on Strongylocentrotus purpuratus populations' newly settled recruits. The genetic analysis of these recruits has been carried out in investigating the hypotheses that the genetic diversity is reduced in the recently settled recruits and that different cohorts are genetically differentiated and the results are reported.