Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

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.

Neurogenic gene regulatory pathways in the sea urchin embryo.

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.

Multicolor labeling in developmental gene regulatory network analysis.

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.

Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos.

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/ß-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/ß-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.

Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos.

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 ß-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.

Rapid adaptation to food availability by a dopamine-mediated morphogenetic response.

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.

The evolution of nervous system patterning: insights from sea urchin development.

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.

Direct development of neurons within foregut endoderm of sea urchin embryos.

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.

TGFß signaling positions the ciliary band and patterns neurons in the sea urchin embryo.

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. TGFß 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 TGFß 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.

The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center.

Two major signaling centers have been shown to control patterning of sea urchin embryos. Canonical Wnt signaling in vegetal blastomeres and Nodal signaling in presumptive oral ectoderm are necessary and sufficient to initiate patterning along the primary and secondary axes, respectively. Here we define and characterize a third patterning center, the animal pole domain (APD), which contains neurogenic ectoderm, and can oppose Wnt and Nodal signaling. The regulatory influence of the APD is normally restricted to the animal pole region, but can operate in most cells of the embryo because, in the absence of Wnt and Nodal, the APD expands throughout the embryo. We have identified many constituent APD regulatory genes expressed in the early blastula and have shown that expression of most of them requires Six3 function. Furthermore, Six3 is necessary for the differentiation of diverse cell types in the APD, including the neurogenic animal plate and immediately flanking ectoderm, indicating that it functions at or near the top of several APD gene regulatory networks. Remarkably, it is also sufficient to respecify the fates of cells in the rest of the embryo, generating an embryo consisting of a greatly expanded, but correctly patterned, APD. A fraction of the large group of Six3-dependent regulatory proteins are orthologous to those expressed in the vertebrate forebrain, suggesting that they controlled formation of the early neurogenic domain in the common deuterostome ancestor of echinoderms and vertebrates.

Gene regulatory network interactions in sea urchin endomesoderm induction.

A major goal of contemporary studies of embryonic development is to understand large sets of regulatory changes that accompany the phenomenon of embryonic induction. The highly resolved sea urchin pregastrular endomesoderm-gene regulatory network (EM-GRN) provides a unique framework to study the global regulatory interactions underlying endomesoderm induction. Vegetal micromeres of the sea urchin embryo constitute a classic endomesoderm signaling center, whose potential to induce archenteron formation from presumptive ectoderm was demonstrated almost a century ago. In this work, we ectopically activate the primary mesenchyme cell-GRN (PMC-GRN) that operates in micromere progeny by misexpressing the micromere determinant Pmar1 and identify the responding EM-GRN that is induced in animal blastomeres. Using localized loss-of -function analyses in conjunction with expression of endo16, the molecular definition of micromere-dependent endomesoderm specification, we show that the TGFbeta cytokine, ActivinB, is an essential component of this induction in blastomeres that emit this signal, as well as in cells that respond to it. We report that normal pregastrular endomesoderm specification requires activation of the Pmar1-inducible subset of the EM-GRN by the same cytokine, strongly suggesting that early micromere-mediated endomesoderm specification, which regulates timely gastrulation in the sea urchin embryo, is also ActivinB dependent. This study unexpectedly uncovers the existence of an additional uncharacterized micromere signal to endomesoderm progenitors, significantly revising existing models. In one of the first network-level characterizations of an intercellular inductive phenomenon, we describe an important in vivo model of the requirement of ActivinB signaling in the earliest steps of embryonic endomesoderm progenitor specification.

A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.

The primary (animal-vegetal) (AV) and secondary (oral-aboral) (OA) axes of sea urchin embryos are established by distinct regulatory pathways. However, because experimental perturbations of AV patterning also invariably disrupt OA patterning and radialize the embryo, these two axes must be mechanistically linked. Here we show that FoxQ2, which is progressively restricted to the animal plate during cleavage stages, provides this linkage. When AV patterning is prevented by blocking the nuclear function of beta-catenin, the animal plate where FoxQ2 is expressed expands throughout the future ectoderm, and expression of nodal, which initiates OA polarity, is blocked. Surprisingly, nodal transcription and OA differentiation are rescued simply by inhibiting FoxQ2 translation. Therefore, restriction of FoxQ2 to the animal plate is a crucial element of canonical Wnt signaling that coordinates patterning along the AV axis with the initiation of OA specification.

Molecular paleoecology: using gene regulatory analysis to address the origins of complex life cycles in the late Precambrian.

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.

The genome of the sea urchin Strongylocentrotus purpuratus.

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.

A database of mRNA expression patterns for the sea urchin embryo.

We present an initial characterization of a database that contains temporal expression profiles of sequences found in 35,282 gene predictions within the sea urchin genome. The relative RNA abundance for each sequence was determined at 5 key stages of development using high-density oligonucleotide microarrays that were hybridized with populations of polyA+ RNA sequence. These stages were two-cell, which represents maternal RNA, early blastula, the time at which major tissue territories are specified, early and late gastrula, during which important morphogenetic events occur, and the pluteus larva, which marks the culmination of pre-feeding embryogenesis. We provide evidence that the microarray reliably reports the temporal profiles for the large majority of predicted genes, as shown by comparison to data for many genes with known expression patterns. The sensitivity of this assay allows detection of mRNAs whose concentration is only several hundred copies/embryo. The temporal expression profiles indicate that 5% of the gene predictions encode mRNAs that are found only in the maternal population while 24% are embryo-specific. Further, we find that the concentration of >80% of different mRNAs is modulated by more than a factor of 3 during development. Along with the annotated sea urchin genome sequence and the whole-genome tiling array (the transcriptome, Samanta, M., Tongprasit, W., Istrrail, S., Cameron, R., Tu, Q., Davidson, E., Stolc, V., in press. A high-resolution transcriptome map of the sea urchin embryo. Science), this database proves a valuable resource for designing experiments to test the function of specific genes during development.

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

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

Tight regulation of SpSoxB factors is required for patterning and morphogenesis in sea urchin embryos.

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 SpKrl. 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.

Patterning the sea urchin embryo: gene regulatory networks, signaling pathways, and cellular interactions.

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.

VEB4: Early zygotic mRNA expressed asymmetrically along the animal-vegetal axis of the 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.