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The segregation of embryonic endomesoderm into separate endoderm and mesoderm fates is not well understood in deuterostomes. Using sea urchin embryos, we showed that Notch signaling initiates segregation of the endomesoderm precursor field by inhibiting expression of a key endoderm transcription factor in presumptive mesoderm. The regulatory circuit activated by this transcription factor subsequently maintains transcription of a canonical Wnt (cWnt) ligand only in endoderm precursors. This cWnt ligand reinforces the endoderm state, amplifying the distinction between emerging endoderm and mesoderm. Before gastrulation, Notch-dependent nuclear export of an essential β-catenin transcriptional coactivator from mesoderm renders it refractory to cWnt signals, insulating it against an endoderm fate. Thus, we report that endomesoderm segregation is a progressive process, requiring a succession of regulatory interactions between cWnt and Notch signaling.

Although it is well established that neural cells are ectodermal derivatives in bilaterian animals, here we report the surprising discovery that some of the pharyngeal neurons of sea urchin embryos develop de novo from the endoderm. The appearance of these neurons is independent of mouth formation, in which the stomodeal ectoderm joins the foregut. The neurons do not derive from migration of ectoderm cells to the foregut, as shown by lineage tracing with the photoactivatable protein KikGR. Their specification and development depend on expression of Nkx3-2, which in turn depends on Six3, both of which are expressed in the foregut lineage. SoxB1, which is closely related to the vertebrate Sox factors that support a neural precursor state, is also expressed in the foregut throughout gastrulation, suggesting that this region of the fully formed archenteron retains an unexpected pluripotency. Together, these results lead to the unexpected conclusion that, within a cell lineage already specified to be endoderm by a well-established gene regulatory network [Peter IS, Davidson EH (2010) Dev Biol 340:188–199], there also operates a Six3/Nkx3-2–dependent pathway required for the de novo specification of some of the neurons in the pharynx. As a result, neuroendoderm precursors form in the foregut aided by retention of a SoxB1-dependent pluripotent state.

The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.

Early development of animal embryos involves establishing axial polarities that specify the anlage of major tissues in a 3-dimensional pattern. Cell fates are specified on this coordinate system through a combination of differential inheritance of maternal regulatory molecules and signaling interactions among cells. Correct patterning of cell fates along the primary axis of the sea urchin embryo depends on tightly regulating the ratio of activities of two nuclear regulatory proteins, SoxB1 and nuclear β–catenin. The latter acts at the top of the gene regulatory network that specifies mesoderm and endoderm and activates, directly or indirectly, signaling by Delta, Wnt8 and Nodal. In contrast, SoxB1 initially accumulates in all nuclei but is progressively eliminated from presumptive mesoderm and endoderm by β-catenin-dependent transcriptional repression and by localized protein turnover, a novel pathway acting downstream of canonical Wnt signaling. A precise temporal program for SoxB1 down regulation is crucial for endomesoderm development because SoxB1 interferes with β–catenin's transcriptional regulatory function. The mechanisms we are beginning to understand that govern the β–catenin-SoxB1 antagonism in sea urchin embryos are likely to have broad significance, since Sox factors are involved in regulating many developmental processes in many deuterostome embryos.

SUMMARY Molecular paleoecology is the application of molecular data to test hypotheses made by paleoecological scenarios. Here, we use gene regulatory analysis to test between two competing paleoecological scenarios put forth to explain the evolution of complex life cycles. The first posits that early bilaterians were holobenthic, and the evolution of macrophagous grazing drove the exploitation of the pelagos by metazoan eggs and embryos, and eventually larvae. The alternative hypothesis predicts that early bilaterians were holopelagic, and new adult stages were added on when these holopelagic forms began to feed on the benthos. The former hypothesis predicts that the larvae of protostomes and deuterostomes are not homologous, with the implication that larval-specific structures, including the apical organ, are the products of convergent evolution, whereas the latter hypothesis predicts homology of larvae, specifically homology of the apical organ. We show that in the sea urchin

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.

During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.

Localization of nuclear β-catenin initiates specification of vegetal fates in sea urchin embryos. We have identified SpKrl, a gene that is activated upon nuclear entry of β-catenin. SpKrl is upregulated when nuclear β-catenin activity is increased with LiCl and downregulated in embryos injected with molecules that inhibit β-catenin nuclear function. LiCl-mediated SpKrl activation is independent of protein synthesis, indicating that SpKrl is a direct target of β-catenin and TCF. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, SpKrl mRNA injection rescues some vegetal structures in β-catenin-deficient embryos. SpKrl negatively regulates expression of the animalizing transcription factor, SpSoxB1. We propose that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities.

To examine whether a BMP signaling pathway functions in specification of cell fates in sea urchin embryos, we have cloned sea urchin BMP2/4, analyzed its expression in time and space in developing embryos and assayed the developmental consequences of changing its concentration through mRNA injection experiments. These studies show that BMP4 mRNAs accumulate transiently during blastula stages, beginning around the 200-cell stage, 14 hours postfertilization. Soon after the hatching blastula stage, BMP2/4 transcripts can be detected in presumptive ectoderm, where they are enriched on the oral side. Injection of BMP2/4 mRNA at the one-cell stage causes a dose-dependent suppression of commitment of cells to vegetal fates and ectoderm differentiates almost exclusively as a squamous epithelial tissue. In contrast, NOGGIN, an antagonist of BMP2/4, enhances differentiation of endoderm, a vegetal tissue, and promotes differentiation of cells characteristic of the ciliated band, which contains neurogenic ectoderm. These findings support a model in which the balance of BMP2/4 signals produced by animal cell progeny and opposing vegetalizing signals sent during cleavage stages regulate the position of the ectoderm/ endoderm boundary. In addition, BMP2/4 levels influence the decision within ectoderm between epidermal and nonepidermal differentiation.

We discuss recent progress in understanding how cell fates are specified along the animal‐vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (b-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear b-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of b-catenin’s transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm‐ ectoderm border within veg1 progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal‐vegetal axis. © 2000 Academic Press

We have identified a Sox family transcription factor, SpSoxB1, that is asymmetrically distributed among blastomeres of the sea urchin embryo during cleavage, beginning at 4th cleavage. SpSoxB1 interacts with a cis element that is essential for transcription of SpAN, a gene that is activated cell autonomously and expressed asymmetrically along the animal-vegetal axis. In vitro translated SpSoxB1 forms a specific complex with this cis element whose mobility is identical to that formed by a protein in nuclear extracts. An anti-SpSoxB1 rabbit polyclonal antiserum specifically supershifts this DNA-protein complex and recognizes a single protein on immunoblots of nuclear proteins that comigrates with in vitro translated SpSoxB1. Developmental immunoblots of total proteins at selected early developmental stages, as well as EMSA of egg and 16-cell stage proteins, show that SpSoxB1 is present at low levels in unfertilized eggs and progressively accumulates during cleavage. SpSoxB1 maternal transcripts are uniformly distributed in the unfertilized egg and the protein accumulates to similar, high concentrations in all nuclei of 4- and 8-cell embryos. However, at fourth cleavage, the micromeres, which are partitioned by asymmetric division of the vegetal 4 blastomeres, have reduced nuclear levels of the protein, while high levels persist in their sister macromeres and in the mesomeres. During cleavage, the uniform maternal SpSoxB1 transcript distribution is replaced by a zygotic nonvegetal pattern that reinforces the asymmetric SpSoxB1 protein distribution and reflects the corresponding domain of SpAN mRNA accumulation at early blastula stage (∼150 cells). The vegetal region lacking nuclear SpSoxB1 gradually expands so that, after blastula stage, only cells in differentiating ectoderm accumulate this protein in their nuclei. The results reported here support a model in which SpSoxB1 is a major regulator of the initial phase of asymmetric transcription of SpAN in the nonvegetal domain by virtue of its distribution at 4th cleavage and is subsequently an important spatial determinant of expression in the early blastula. This factor is the earliest known spatially restricted regulator of transcription along the animal-vegetal axis of the sea urchin embryo.

Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis of the very early sea urchin embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71-78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271-1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a β-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the animal-vegetal axis of the sea urchin embryo.

We have used the Xenopus embryo as a test system for analyzing the activity of SpAN, a sea urchin metalloprotease in the astacin family containing BMP1 and tolloid. Embryos expressing SpAN initiated gastrulation on a time scale indistinguishable from controls, but invagination of the vegetal pole was subsequently delayed by several hours. At tailbud stages the most severely affected embryos were completely ventralized, lacking all dorsal structures. Molecular analysis of injected embryos, using probes for both dorsal (xgsc and xnot) and ventral (xhox3 and xwnt8) mesoderm, indicates that SpAN ventralizes dorsal mesoderm during gastrula stages. These results mirror those previously obtained with BMP4, suggesting that SpAN may enhance the activity of this ventralizing factor. Consistent with this suggestion, we have shown that SpAN blocks the dorsalizing activity of noggin and chordin, two inhibitory binding proteins for BMP4, but not that of a dominant-negative receptor for BMP4. In contrast, a dominant-negative SpAN, in which the metalloprotease domain has been deleted, dorsalizes ventral mesoderm, a phenotype that can be rescued by coexpressing either SpAN or XBMP1. This suggests that SpAN is mimicking a Xenopus metalloprotease responsible for regulating the activity of Xenopus BMPs during gastrulation. Moreover, our results raise the possibility that SpAN may function to facilitate BMP signaling in early sea urchin embryos.

We have shown previously by in situ hybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoon et al., J. Biol. Chem. 271, 20119-20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find that SpFGFR transcripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive, SpFGFR transcripts in lantern muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences.

The sea urchin embryo is an important model system for developmental gene regulatory network (GRN) analysis. This chapter describes the use of multicolor fluorescent in situ hybridization (FISH) as well as a combination of FISH and immunohistochemistry in sea urchin embryonic GRN studies. The methods presented here can be applied to a variety of experimental settings where accurate spatial resolution of multiple gene products is required for constructing a developmental GRN.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus . The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

We have analyzed a gene, designated VEB4, that is expressed transiently in very early blastulae of the sea urchin, Strongylocentrotus purpuratus. Sequence analysis of the complete open reading frame shows that VEB4 encodes an unusual, highly charged protein with a pl of 9.55. We show here that VEB4 mRNA accumulate in a spatial pattern that is indistinguishable from that of two other recently described genes encoding metallo-endoproteases, SpAN, related to astacin and SpHE, the hatching enzyme (Reynolds et al. 1992). VEB4 and other members of this gene set encode the earliest strictly zygotic gene products that have been identified. The asymmetric accumulation of VEB4 mRNA in non-vegetal blastomeres of the 16 cell embryo and their descendants reflects the animal-vegetal maternal developmental axis.

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other’s activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.

Specification of cell fate in sea urchin embryos involves initial asymmetric distribution of maternal molecules that establish vegetal and nonvegetal domains of transcription activity. Subsequently, fates of most blastomeres are regulated by cell–cell interactions involving signalling ligands and cell surface receptors. Keywords: maternal determinants; cell–cell interactions; fate specification; transcription factors; receptors

Recent studies of the sea urchin embryo have elucidated the mechanisms that localize and pattern its nervous system. These studies have revealed the presence of two overlapping regions of neurogenic potential at the beginning of embryogenesis, each of which becomes progressively restricted by separate, yet linked, signals, including Wnt and subsequently Nodal and BMP. These signals act to specify and localize the embryonic neural fields – the anterior neuroectoderm and the more posterior ciliary band neuroectoderm – during development. Here, we review these conserved nervous system patterning signals and consider how the relationships between them might have changed during deuterostome evolution.

between these genes, we have found that transcriptional feedbackloops acting within and between adjacent cells are important inestablishing the cell type pattern. Specifically, the WER MYB-typeprotein, the GL3/EGL3 bHLH-type proteins, and the TTG WD-proteinappear to interact in a transcriptional complex to positively regulatethe GL2, CPC, TRY, and ETC1 genes. The GL2 homeodomaintranscription factor is involved in regulating genes that generatethe non-hair cell type. The CPC, TRY, and ETC1 proteins arestructurally-related small MYB transcription factors that appear tomove between cells and inhibit the formation of the WER-GL3/EGL3-TTG complex; this negative regulation represents a type of lateralinhibition mechanism. The position-dependent pattern relies on aleucine-rich-repeat receptor-like kinase (SCRAMBLED (SCM)), thatappears to influence WER gene expression causing an unequaldistribution of the transcriptional regulators in the N and H cellpositions. Current research is focused on using genomics, bioinfor-matics, and math modeling to further dissect the regulatory network.These studies are likely to provide new insights into the basicmechanisms of regulatory gene networks and cell-type specificationduring development.doi:10.1016/j.ydbio.2011.05.056Program/Abstract # 40Neurons develop in situ in foregut endoderm of seaurchin embryosZheng Wei

We have determined the expression pattern of arylsulfatase in embryos of the sea urchin Strongylocentrotus purpuratus. Polyclonal antibodies raised against a fusion protein containing sequences encoded by SpARSI (Yang et al., 1989, Dev. Biol. 135: 53–61, 1989) detect several peptides of 65–70 kD on immunoblots. Treatment with glycopeptidase F shows that at least one of these peptides is modified by N-linked glycosylation, which accounts for some of the peptide diversity. We have also identified a second arylsulfatase gene (SpARSII) whose sequence is highly similar to ARS, a gene expressed in the Hemicentrotus pulcherrimus embryo. Arylsulfatase activity is detectable in unfertilized eggs, in which only SpARSII mRNA can be detected. Both SpARSI and SpARSII mRNAs increase greatly in abundance during embryogenesis accompanied by parallel changes in arylsulfatase activity and immunoreactivity. Immunohistochemistry with the anti-SpARSI antibody shows that arylsulfatase accumulates primarily along the apical surface of the aboral ectoderm of pluteus larvae, and to a lesser extent along portions of oral ectoderm. At earlier stages, the protein is more uniformly distributed along all presumptive ectoderm, reflecting a more uniform mRNA distribution. Treatment of embryos with glycine-EDTA, which dissociates but does not lyse cells of the embryo, releases virtually all enzymatic activity and all immunoreactive protein. Embryos cultured in sulfate-free sea water, which arrest at gastrula stage, show normal accumulation and secretion of peptide detected with the SpARSI antibody.

The cloning and characterization of cDNAs representing four genes or small gene families that are coordinately expressed in a spatially restricted pattern during the very early blastula (VEB) stage of sea urchin development are presented. The VEB genes encode multiple transcripts that are expressed transiently in embryos of Strongylocentrotus purpuratus between 16-cell stage and hatching, with peak abundance 12 to 15 hours postfertilization (∼150-250 cells). The VEB transcripts share the same spatial pattern in the early blastula embryo: they are asymmetrically distributed along the animalvegetal axis but their distribution around this axis is uniform. Thus, the VEB transcripts are the earliest messages to reveal asymmetry along the primary axis in the sea urchin embryo. The temporal and spatial patterns of VEB transcript accumulation are not consistent with involvement of these gene products in cell division or in tissue-specific functions. Furthermore, VEB messages cannot be detected in either ovary or adult tissues, suggesting that these genes function exclusively during embryogenesis. We suggest that the VEB genes function in constructing the early blastula. Two VEB genes encode metalloendoproteases: one (SpHE) is hatching enzyme and the other (SpAN) is similar to bone morphogenetic protein-1 (BMP-1; Wozney et al., Science 242: 1528-1534, 1988) and the Tolloid gene product (tld) (Shimell et al., Cell 67: 459482, 1991). Several lines of evidence suggest that the VEB genes are regulated directly by factors or regulatory activities localized along the maternally specificed animal-vegetal axis.

During development of the sea urchin Strongylocentrotus purpuratus embryo, transcription of the Sped and actin Cyllla genes is activated and the corresponding mRNAs accumulate specifically in ectoderm cells. We show that in gastrulae this tissue specificity of mRNA accumulation is regulated largely if not entirely at a posttranscriptional level. We used RNAase protection assays with intron and exon probes to measure the levels of nuclear precursors and mature message, respectively, in total RNA from embryo fractions enriched for ectoderm (Ect) or endoderm + mesenchyme (E/M) cells. These measurements demonstrate that E/M cells, which do not accumulate Sped and actin Cyllla mRNAs, contain high levels of intron transcripts, indicating that cells of the E/M tissues transcribe these genes. At later stages, transcripts containing intron sequences are restricted to ectoderm cells. These results indicate that there is a transition from posttranscriptional to transcriptional regulation of tissue-specific mRNA accumulation during the gastrula stage. Measurements of transcription rate by nuclear run-on assays substantiate this conclusion for Sped and extend it to two other genes, SpEGFI and Spec2c, which also encode ectoderm-specific mRNAs. Posttranscriptional regulation was not observed for the SM50 gene whose mRNA accumulates only in primary mesenchyme cells, or for actin Cyl which is expressed predominantly in E/M cells of gastrulae.

Summary: This article describes the use of in situ hybridization to study gene expression. We consider the advantages of this technique in comparison to other methods for analyzing gene expression at the level of messenger RNAs, and compare the information that can be obtained by localization of mRNAs by in situ hybridization versus localization of proteins by immunohistochemistry. We describe applications of in situ hybridization to the study of normal and abnormal development and tissue function. We then present the protocol developed in our laboratory as a framework for detailed discussion of each of the steps in the procedure, and comment on the relative merits of different options at each step. Individual steps discussed include tissue fixation and prehybridization treatments, choice of probes, hybridization parameters (probe concentration, time, and temperature), posthybridization washes, choice of isotopes and nonisotopic methods for detecting hybridized probe, and autoradiography. Finally,...

Many proteins containing domains related to epidermal growth factor (EGF) function in intercellular interactions that mediate specification of cell fate. We have used in situ hybridization to show that the expression of two EGF-related genes (SpEGF I and SpEGF II) is restricted to the same subset of ectodermal cells in sea urchin pluteus larvae. However, the concentration of EGF I mRNA in different epithelial cells of aboral ectoderm and postoral facial epithelium is constant while that of EGF II mRNA is highly modulated. RNase protection assays show that both genes are activated during the period when ectoderm founder cells are established, i.e., between fourth and fifth and between fifth and sixth cleavages for EGF I and EGF II, respectively. By mesenchyme blastula stage EGF I mRNA reaches maximum abundance (800–1000 copies/expressing cell) as a result of a high transcription rate, while EGF II mRNA peaks at about half that concentration by gastrula stage. EGF I expression begins at early stages of oogenesis while EGF II expression appears to be confined to embryogenesis.

Molecular paleoecology is the application of molecular data to test hypotheses made by paleoecological scenarios. Here, we use gene regulatory analysis to test between two competing paleoecological scenarios put forth to explain the evolution of complex life cycles. The first posits that early bilaterians were holobenthic, and the evolution of macrophagous grazing drove the exploitation of the pelagos by metazoan eggs and embryos, and eventually larvae. The alternative hypothesis predicts that early bilaterians were holopelagic, and new adult stages were added on when these holopelagic forms began to feed on the benthos. The former hypothesis predicts that the larvae of protostomes and deuterostomes are not homologous, with the implication that larval-specific structures, including the apical organ, are the products of convergent evolution, whereas the latter hypothesis predicts homology of larvae, specifically homology of the apical organ. We show that in the sea urchin, Strongylocentrotus purpuratus, the transcription factors NK2.1 and HNF6 are necessary for the correct spatial expression profiles of five different cilia genes. All of these genes are expressed exclusively in the apical plate after the mesenchyme-blastula stage in cells that also express NK2.1 and HNF6. In addition, abrogation of SpNK2.1 results in embryos that lack the apical tuft. However, in the red abalone, Haliotis rufescens, NK2.1 and HNF6 are not expressed in any cells that also express these same five cilia genes. Nonetheless, like the sea urchin, the gastropod expresses both NK2.1 and FoxA around the stomodeum and foregut, and FoxA around the proctodeum. As we detected no similarity in the development of the apical tuft between the sea urchin and the abalone, these molecular data are consistent with the hypothesis that the evolution of mobile, macrophagous metazoans drove the evolution of complex life cycles multiple times independently in the late Precambrian.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus , a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Publisher Summary The chapter discusses the properties of morpholinos and the reasons behind their having significant advantages over other antisense and loss-of-function approaches. Current methods for using morpholinos and for analyzing their effects on the development of basal deuterostome embryos are described. The chapter also reviews the kinds of information that are gained from loss-of-function morpholino approaches and considers other possible applications of this technology. The most important approaches for determining the function of genes during development are either mis/overexpression or loss-of-function assays. In lower deuterostome embryos—principally, the sea urchins and the tunicates—the use of morpholinos has revolutionized the analysis of gene function during development. In addition to demonstrating the developmental roles of individual genes, this approach has allowed investigations of the epistatic relationships among gene regulatory factors and signaling pathways, which has led to the elucidation of gene regulatory networks that control cell fate specification and differentiation. The mechanism of morpholino-mediated antisense interference is fundamentally different from that of other antisense approaches. It does not require degradation of target mRNAs by cellular RNase H activity but instead physically blocks translation or access to the splicing apparatus.

We discuss steps in the specification of major tissue territories of the sea urchin embryo that occur between fertilization and hatching blastula stage and the cellular interactions required to coordinate morphogenetic processes that begin after hatching. We review evidence that has led to new ideas about how this embryo is initially patterned: (1) Specification of most of the tissue territories is not direct, but proceeds gradually by progressive subdivision of broad, maternally specified domains that depend on opposing gradients in the ratios of animalizing transcription factors (ATFs) and vegetalizing (beta-catenin) transcription factors; (2) the range of maternal nuclear beta-catenin extends further than previously proposed, that is, into the animal hemisphere, where it programs many cells to adopt early aboral ectoderm characteristics; (3) cells at the extreme animal pole constitute a unique ectoderm region, lacking nuclear beta-catenin; (4) the pluripotential mesendoderm is created by the combined outputs of ATFs and nuclear beta-catenin, which initially overlap in the macromeres, and by an undefined early micromere signal; (5) later micromere signals, which activate Notch and Wnt pathways, subdivide mesendoderm into secondary mesenchyme and endoderm; and (6) oral ectoderm specification requires reprogramming early aboral ectoderm at about the hatching blastula stage. Morphogenetic processes that follow initial fate specification depend critically on continued interactions among cells in different territories. As illustrations, we discuss the regulation of (1) the ectoderm/endoderm boundary, (2) mesenchyme positioning and skeletal growth, (3) ciliated band formation, and (4) several suppressive interactions operating late in embryogenesis to limit the fates of multipotent cells.

We discuss steps in the specification of major tissue territories of the sea urchin embryo that occur between fertilization and hatching blastula stage and the cellular interactions required to coordinate morphogenetic processes that begin after hatching. We review evidence that has led to new ideas about how this embryo is initially patterned: (1) Specification of most of the tissue territories is not direct, but proceeds gradually by progressive subdivision of broad, maternally specified domains that depend on opposing gradients in the ratios of animalizing transcription factors (ATFs) and vegetalizing (beta-catenin) transcription factors; (2) the range of maternal nuclear beta-catenin extends further than previously proposed, that is, into the animal hemisphere, where it programs many cells to adopt early aboral ectoderm characteristics; (3) cells at the extreme animal pole constitute a unique ectoderm region, lacking nuclear beta-catenin; (4) the pluripotential mesendoderm is created by the combined outputs of ATFs and nuclear beta-catenin, which initially overlap in the macromeres, and by an undefined early micromere signal; (5) later micromere signals, which activate Notch and Wnt pathways, subdivide mesendoderm into secondary mesenchyme and endoderm; and (6) oral ectoderm specification requires reprogramming early aboral ectoderm at about the hatching blastula stage. Morphogenetic processes that follow initial fate specification depend critically on continued interactions among cells in different territories. As illustrations, we discuss the regulation of (1) the ectoderm/endoderm boundary, (2) mesenchyme positioning and skeletal growth, (3) ciliated band formation, and (4) several suppressive interactions operating late in embryogenesis to limit the fates of multipotent cells.

Differentiation of sea urchin embryo ectoderm, endoderm and mesenchyme cells, whose anlagen are arrayed along the animal-vegetal axis, relies on both maternally regulated localized transcription factor activities and cell-cell signalling. Classic models proposed that fates are determined by opposing animal and vegetal morphogenetic gradients, whereas current models emphasize unidirectional and sequential vegetal-to-animal signalling cascades between adjacent blastomeres. Recent data support aspects of both models: the vegetal micromeres send one or more signals, which depend on a nuclear beta-catenin-dependent pathway, that both activate Notch signalling required for secondary mesenchyme fate and promote endoderm differentiation and gastrulation. This is opposed by an animalizing domain of BMP4 signals that regulates ectodermal cell fates and establishes the ectoderm-endoderm border.

We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula. The Strongylocentrotus purpuratus hatching enzyme gene, SpHE, is transcribed transiently only in nonvegetal blastomeres during the cleavage and very early blastula stages of sea urchin development (20). The activation of SpHE is early and cell autonomous and therefore is very likely to be regulated by localized maternal transcription-regulatory activities partitioned asymmetrically along the animal-vegetal (AV) axis. Consequently, the SpHE gene represents an excellent entry point for determining how the AV axis is established, through the identification of trans-acting factors that regulate its transcription. Previously, we reported that a relatively compact region of the SpHE promoter, consisting of 300 bp upstream of the transcription initiation site, is sufficient to sponsor both highlevel transcriptional activity and correct nonvegetal spatial expression (26, 27). Although this regulatory region is small, nine cis-acting elements have been defined within it, which can form complexes with at least six different proteins, as shown by in vitro DNase I footprinting and electrophoretic mobility shift assays (EMSAs) (26). Most of these cis elements were found to be occupied when the gene is active in vivo but to be unoccupied and in a nucleosome-like configuration when the gene is inactive (28). Extensive mutational dissection of the SpHE regulatory region supports a model in which all of the DNA binding proteins confer positive function in nonvegetal blastomeres (26, 27). This suggests that an important mechanism establishing AV polarity of developmental potential in the early sea urchin embryo is partitioning of positive maternal regulatory activities to nonvegetal blastomeres. To test this model, we have begun to investigate the SpHE transcriptional regulators. Here we report the cloning of an S. purpuratus egg cDNA encoding a member of the Ets family, using a nontraditional yeast one-hybrid genetic screening approach in which a large portion of the SpHE promoter sequence instead of individual multimerized cis elements was used as bait. Using this method, we isolated 13 strong positive clones containing overlapping segments of the same sequence, which encodes a member of the Ets transcription factor family. Because this protein contains a conserved Ets domain that is much more similar to that of Drosophila Ets4 (6) than to any other Ets domain, we have named it SpEts4. We demonstrate that the SpEts4 gene sequence encodes a protein that binds with the same specificity, and forms a complex of similar mobility in EMSA, as does the native protein found in nuclear protein extracts from very early blastulae. Using SpHE promoter transgenes, we show that the site to which SpEts4 binds confers positive regulatory activity and that exogeneously supplied recombinant SpEts4 augments promoter activity. Consistent with a positive function for SpEts4 in regulating SpHE transcription, SpEts4 transcripts accumulate in the egg and early embryo transiently and just prior to the burst of SpHE mRNA expression.

Previous studies of the regulatory region of the SpHE (hatching enzyme) gene of the sea urchin Strongylocentrotus purpuratus (Wei, Z., Angerer, L.M., Gagnon, M.L. and Angerer, R.C. (1995) Characterization of the SpHE promoter that are spatially regulated along the animal-vegetal axis of the sea urchin embryo. Dev. Biol. 171, 195–211) have shown that approximately 330 bp is necessary and sufficient to promote high level expression in embryos of transgenes that reproduce the spatially asymmetric pattern of endogenous gene activity along the maternally determined animal-vegetal embryonic axis. Furthermore, SpHE regulatory elements appear to be redundant since several different combinations are sufficient to elicit strong promoter activity and many subsets function like the endogenous gene only in non-vegetal cells of the blastula (Wei, Z., Angerer, L.M. and Angerer, R.C. (1997) Multiple positive cis -elements regulate the asymmetric expression of the SpHE gene along the sea urchin embryo animal-vegetal axis. Dev. Biol., 187, 71–88). Here we demonstrate by in vivo footprinting that many cis elements on the endogenous promoter are occupied when the gene is active in early blastulae, but the binding of corresponding trans factors is significantly reduced when the gene becomes inactive in late blastulae. In addition, downregulation of the promoter is accompanied by a transition from a non-nucleosomal to a nucleosome-like chromatin structure. Surprisingly, in vitro DNase I footprints of the 300 bp promoter using nuclear protein extracts from early and late blastulae are not detectably different and neither this sequence, nor a longer one extending to −1255, reproduces the loss of endogenous SpHE transcriptional activity after very early blastula stage. These observations imply that temporal repression of SpHE transcription involves a decrease in accessibility of the promoter to activators that are nevertheless present in nuclei and capable of activating transgene promoters. Temporal, but not spatial, downregulation is therefore likely to be regulated by negative activities functioning outside the −1255 promoter region which may serve as direct repressors or mediate an inactive chromatin structure.

The mechanism that establishes the maternally determined animal–vegetal axis of sea urchin embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.

ROBERT C. ANGERER AND LYNNE M. ANGERER Department QfBiology, University afRochester, Rochester, New York 14627 Introduction Like those of a large majority of taxa, sea urchin em- bryos establish a spatial coordinate system for the initial body plan from one axis, the animal-vegetal (A-V), that is fixed during oogenesis by asymmetric deposition of maternal molecules (the embryologists’ “determinants”) and a second axis, dorsal-ventral (or, more descriptively, oral-aboral), that is specified sometime during the first few cleavage divisions (reviewed by Davidson, 1989). The ability of sea urchin embryos to establish these axes while continuously reorienting in culture suggests that neither axis is sensitive to the earth’s gravitational field. In embryos of many sea urchin species, A-V polarity is evidenced by the unequal sizes of blastomeres of the 16- cell embryo, which consists of tiers of eight mesomeres, four macromeres, and four micromeres. Classical exper- imental micromanipulations of embryos (reviewed by Horstadius, 1973) have established that the fates of mi- cromeres are determined by inheritance of maternal molecules. In addition, the micromeres provide a vegetal focus of inductive influence that is critical in the normal embryo for appropriate specification of fates of overlying animal blastomeres, and that can induce vegetal differ- entiation (gut, secondary mesenchyme) in cells of more animal tiers when micromeres are transplanted to ec- topic sites (Khaner and Wilt, 199 1; Ransick and David- son, 1993). Thus, specification of fates along the AV axis utilizes both major mechanisms familiar to developmen- tal biologists-inheritance of maternally provided posi

We describe the cloning of a new fibroblast growth factor receptor, SpFGFR1, that is differentially regulated at the level of transcript abundance during sea urchin embryogenesis. Sequence representing the conserved tyrosine kinase domain was obtained by reverse transcription-polymerase chain reaction using degenerate primers, and the entire open reading frame was obtained by standard cDNA library screening methods. SpFGFR contains a series of domains characteristic of FGFRs: three immunoglobulin-like motifs, an acid box, a transmembrane domain, a relatively long juxtamembrane sequence, a split tyrosine kinase domain, and two conserved intracellular tyrosine residues. Alternative splicing of SpFGFR generates two variants (Ig3L and Ig3S), which differ by insertion in the center of the Ig3 domain of 34 extra amino acids, encoded by an additional exon. Transcripts encoding both variants accumulate when morphogenesis begins with mesenchyme cell ingression and gastrulation. SpFGFR transcripts accumulate in all cell types of the embryo, although in situ hybridization shows that they are somewhat enriched in cells of oral ectoderm and endoderm. Transcripts encoding the Ig3S variant, whose structure resembles more closely that of vertebrate receptors, are enriched in endomesoderm, suggesting that the SpFGFR variants could play distinct roles in the sea urchin embryo.

In order to investigate how the maternally specified animal-vegetal axis of the sea urchin embryo is established, we have examined the molecular basis of regulation of several genes transcribed differentially in nonvegetal and vegetal domains of the very early blastula. Here we present an initial characterization of the regulatory region of one of these, SpAN, which encodes a protease in the astacin family related to Drosophila tolloid and vertebrate BMP-1 (Reynolds et al., Development 114, 769-786). Tests of SpAN promoter function in vivo show that high-level activity and correct not-vegetal expression are mediated by sequences within 300 bp upstream of the basal promoter. In vitro studies have identified six protein binding sites serviced by at least five different proteins. Comparison of the structure of the SpAN promoter to that of SpHE, whose expression pattern is identical, shows that both promoters contain multiple positively acting upstream elements close to the basal promoter. We show that two elements are critical for high-level transcription of SpAN, since exact replacement of either results in 10- to 20-fold reduction in promoter strength. These shared elements are, however, not essential for spatially correct SpHE gene transcription. We conclude that the coordinate strong activities of the SpAN and SpHE promoters in the nonvegetal domain of the embryo rely primarily on different transcription factor activities.

Three different Wnt signaling pathways function to restrict the anterior neuroectoderm state to the anterior end of the sea urchin embryo, a mechanism of anterior fate restriction that could be conserved among deuterostomes., Patterning the neuroectoderm along the anterior–posterior (AP) axis is a critical event in the early development of deuterostome embryos. However, the mechanisms that regulate the specification and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same transcription factors and secreted modulators of Wnt signaling, as does the early vertebrate ANE (forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the anterior end of the embryo requires a Wnt/β-catenin-dependent signaling mechanism. Here we use morpholino- or dominant negative-mediated interference to demonstrate that the early sea urchin embryo integrates information not only from Wnt/β-catenin but also from Wnt/Fzl5/8-JNK and Fzl1/2/7-PKC pathways to provide precise spatiotemporal control of neuroectoderm patterning along its AP axis. Together, through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of re-specification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the most anterior blastomeres. There, the Wnt receptor antagonist, Dkk1, protects this state through a negative feedback mechanism. Because these different Wnt pathways converge on the same cell fate specification process, our data suggest they may function as integrated components of an interactive Wnt signaling network. Our findings provide strong support for the idea that the sea urchin ANE regulatory state and the mechanisms that position and define its borders represent an ancient regulatory patterning system that was present in the common echinoderm/vertebrate ancestor., Author Summary The initial regulatory state of most cells in many deuterostome embryos, including those of vertebrates and sea urchins, supports anterior neural fate specification. It is important to restrict this neurogenic potential to the anterior end of the embryo during early embryogenesis, but the molecular mechanisms by which this re-specification of posterior fate occurs are incompletely understood in any embryo. The sea urchin embryo is ideally suited to study this process because, in contrast to vertebrates, anterior–posterior neuroectoderm patterning occurs independently of dorsal-ventral axis patterning and takes place before the complex cell movements of gastrulation. In this study, we show that a linked, three-step process involving at least three different Wnt signaling pathways provides precise spatiotemporal restriction of the anterior neuroectoderm regulatory state to the anterior end of the sea urchin embryo. Because these three pathways impinge on the same developmental process, they could be functioning as an integrated Wnt signaling network. Moreover, striking parallels among gene expression patterns and functional studies suggest that this mechanism of anterior fate restriction could be highly conserved among deuterostomes.

We have identified a gene in the sea urchin Strongylocentrotus purpuratus that encodes a member of the transforming growth factor β (TGF-β) gene superfamily. We have named the gene univin, and it is the first member of this superfamily to be reported in echinoderms. The cDNA sequence predicts a 383-amino-acid residue protein with 7 cysteine residues characteristic of members of this superfamily and with a cluster of basic residues appropriately situated to signal proteolytic cleavage. Sequence comparisons place univin in the bone morphogenetic protein (BMP) group of the TGF-β superfamily along with the vertebrate BMPs, decapentaplegic protein from Drosophila, and Vg-1 from Xenopus. Analyses of univin expression in early embryos by RNA blots and in situ hybridization revealed the highest levels of expression in the egg and prehatching blastula. During late cleavage stages, univin mRNA accumulation is progressively restricted to a circumequatorial band. Expression is further restricted during gastrulation when univin transcripts are detected primarily in the presumptive foregut and ciliated band. By pluteus stage, signals are detectable only in these cell types. The restricted temporal and spatial patterns of expression of univin during early blastula stages parallel those of SpAN , which encodes an astacin-like protease related to tolloid and BMP-1 (Reynolds et al., 1992). The fact that these proteases are thought to function in the proteolytic activation of TGF-β-related proteins that, respectively, regulate Drosophila embryonic dorsal-ventral patterning and vertebrate bone development suggests that SpAN and univin could also have critical roles in early developmental decisions in the sea urchin embryo.

Cloning and sequencing of Tripneustes gratilla genomic DNA and cDNA encoding a developmentally regulated, embryonic messenger RNA, referred to as Tg616, revealed an actin-encoding gene orthologous to the CyI actin gene described from Strongylocentrotus purpuratus. Tg616 and SpCyI share: (1) 150 nucleotides of highly conserved sequence 5′ of the transcription start site, (2) 95% nucleotide sequence identity in the protein encoding regions, which specify identical amino acid residues in 375 of 377 positions, and (3) extensive nucleotide sequence identity in the 3′ untranslated region of their messenger RNAs. Tg616 was therefore designated TgCyI. In situ hybridization shows sequential activation of TgCyI in various cells of the embryo. TgCyI mRNA becomes abundant in primary and secondary mesenchyme cells as they prepare to enter the blastocoel, in prospective aboral ectoderm cells at blastula stage, in gut cells during gut differentiation, and in oral ectoderm at pluteus stage. This pattern of embryonic gene expression is more complex than any of the major patterns of developmentally upregulated genes observed in S. purpuratus embryos and is distinct from SpCyI expression which is progressively restricted to the gut and oral ectoderm.

Food can act as a powerful stimulus, eliciting metabolic, behavioural and developmental responses. These phenotypic changes can alter ecological and evolutionary processes; yet, the molecular mechanisms underlying many plastic phenotypic responses remain unknown. Here we show that dopamine signalling through a type-D(2) receptor mediates developmental plasticity by regulating arm length in pre-feeding sea urchin larvae in response to food availability. Although prey-induced traits are often thought to improve food acquisition, the mechanism underlying this plastic response acts to reduce feeding structure size and subsequent feeding rate. Consequently, the developmental programme and/or maternal provisioning predetermine the maximum possible feeding rate, and food-induced dopamine signalling reduces food acquisition potential during periods of abundant resources to preserve maternal energetic reserves. Sea urchin larvae may have co-opted the widespread use of food-induced dopamine signalling from behavioural responses to instead alter their development.

We have investigated the temporal and spatial patterns of accumulation of mRNAs randomly selected from the sea urchin gastrula polyadenylated RNA population. Three different assays show that the predominant temporal pattern of expression, exhibited by about three-fourths of these messages, consists of a large (mean 80-fold) increase in mRNA abundance between egg and gastrula stages. Most mRNAs are present in the maternal population and are detectable on blots as single mature-sized messages; however, a large number of high-molecular-weight, heterodisperse transcripts containing these same sequences also exist in the egg cytoplasm. The majority of gastrula messages are not embryo specific but are present in total adult urchin RNA at concentrations similar to those in embryos. Fine-scale RNA blot analysis indicates that the majority of mRNAs begin to accumulate at very early blastula stages, although there is considerable diversity in the time when these messages reach peak abundance. Most gastrula mRNAs are also spatially regulated during development. The observed distributions can be categorized into three major functional or regulatory classes: (1) Forty percent of mRNAs accumulate in cells which are cycling or preparing for growth. (2) About one-third of the messages accumulate in one or more differentiating cell types. (3) Only slightly more than one-fourth of the messages are present in all cell types throughout development. Most tissue-specific messages are relatively abundant, indicating that the differentiated functions of cells are executed through mRNAs operating at the level of hundreds of copies per cell. In contrast, most rare messages are expressed in most or all cell types, in which they function at only a few copies per cell. All messages which begin to accumulate before hatching blastula stage are initially distributed broadly, and their distribution becomes progressively restricted during embryogenesis. In contrast, all messages which begin to accumulate after the onset of gastrulation accumulate only in discrete subsets of cells. The results presented here illustrate much more extensive temporal regulation of gene expression during sea urchin embryogenesis than previously detected. This is accompanied by spatial regulation of expression of most genes which is itself temporally modulated as the cellular requirements for cell division and differentiation change during development.

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.