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.

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.

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).

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.

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.

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.

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.

TGFβ 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. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Immunochemical Analysis of Arylsulfatase Accumulation in Sea Urchin Embryos: (extracellular matrix/arylsulfatase/sea urchin embryo/tissue-specific gene products/sea urchin embryo/in situ hybridization).

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.