T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.     

Micromere descendants at the blastula stage are involved in normal archenteron formation in sea urchin embryos

Several lines of evidence suggest that micromere signaling plays a key role in endo-mesoderm differentiation along the animal-vegetal (A-V) axis in sea urchin embryos. A recent study has suggested that the activity of micromeres of inducing endoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula stage in the echinoids Scaphechinus mirabiris and Hemicentrotus pulcherrimus. In the present study, to confirm the inductive capacity of the micromere descendants in normal development, the timing of initiation of gastrulation and the elongation rate of the archenteron were examined in both micromereless embryos and in micromereless embryos cultured until the hatching blastula stage and then recombined with micromere descendants of the same age. The micromereless embryos consistently exhibited a delay in the initiation of gastrulation and a decrease in elongation rate of the archenteron, as compared with those in controls. In contrast, when the micromereless embryos cultured until the hatching blastula stage were recombined with micromere descendants of the same age, the recombinant embryos exhibited rescue of both the delay in initiation of gastrulation and a decrease in elongation rate of the archenteron. The delayed expression of alkaline phosphatase activity, an endoderm-specific marker, in the micromereless embryos was also rescued in the recombinant embryos. The recombined micromere descendants formed the larval spicules in the same schedule as that observed in the controls. These results indicate that at the hatching blastula stage, micromere descendants emanate a signal(s) required for normal gastrulation of the presumptive endo-mesodermal region.     

HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

The mechanism of micromere specification is one of the central issues in sea urchin development. In this study we have identified a sea urchin homologue of ets 1 + 2. HpEts, which is maternally expressed ubiquitously during the cleavage stage and which expression becomes restricted to the skeletogenic primary mesenchyme cells (PMC) after the hatching blastula stage. The overexpression of HpEts by mRNA injection into fertilized eggs alters the cell fate of non-PMC to migratory PMC. HpEts induces the expression of a PMC-specific spicule matrix protein, SM50, but suppresses of aboral ectoderm-specific arylsulfatase and endoderm-specific HpEndo16. The overexpression of dominant negative delta HpEts which lacks the N terminal domain, in contrast, specifically represses SM50 expression and development of the spicule. In the upstream region of the SM50 gene there exists an ets binding site that functions as a positive cis-regulatory element. The results suggest that HpEts plays a key role in the differentiation of PMCs in sea urchin embryogenesis.     

LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties

Signals from micromere descendants play a critical role in patterning the early sea urchin embryo. Previous work demonstrated a link between the induction of mesoderm by micromere descendants and the Notch signaling pathway. In this study, we demonstrate that these micromere descendants express LvDelta, a ligand for the Notch receptor. LvDelta is expressed by micromere descendants during the blastula stage, a time when signaling has been shown to occur. By a combination of embryo microsurgery, mRNA injection and antisense morpholino experiments, we show that expression of LvDelta by micromere descendants is both necessary and sufficient for the development of two mesodermal cell types, pigment cells and blastocoelar cells. We also demonstrate that LvDelta is expressed by macromere descendants during mesenchyme blastula and early gastrula stages. Macromere-derived LvDelta is necessary for blastocoelar cell and muscle cell development. Finally, we find that expression of LvDelta is sufficient to endow blastomeres with the ability to function as a vegetal organizing center and to coordinate the development of a complete pluteus larva.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins,is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through beta-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein.     

Regionalized Cell Division during Sea Urchin Gastrulation Contributes to Archenteron Formation and Is Correlated with the Establishment of Larval Symmetry

During gastrulation of the sea urchin, Lytechinus variegutus there is localized proliferation of cells in the vegetal plate region prior to its invagination. Cell counts show that during gastrulation the number of cells per embryo increases 60% from 1025 to 1640. Measurements of cell volumes suggest that some growth may follow these divisions. Feulgen staining shows that the greatest mitotic activity throughout gastrulation occurs in the vegetal plate region. Labelling embryos with 3H-thymidine reveals that incorporation in the vegetal plate is confined to cells that encircle the base of the archenteron. Pulse-chase experiments indicate that these labelled cells contribute descendants to the vegetal half of the archenteron. Additionally, 3-dimensional reconstructions of vegetal regions at different stages reveal that by the end of gastrulation two bilateral clusters of labelled cells lie at the future sites of the post-oral arms of the pluteus larva, thus marking the axes of bilateral and dorso-ventral symmetry. Our findings suggest that two of the principal events of sea urchin gastrulation — the formation of the archenteron and the establishment of symmetry in the larva — are accompanied by distinct patterns of cell division.     

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 TGFβ 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.     

Characterization and expression of two matrix metalloproteinase genes during sea urchin development

Matrix metalloproteinases (MMPs) play an essential role in a variety of processes in development that require extracellular matrix remodeling and degradation. In this study, we characterize two MMPs from the sea urchin Strongylocentrotus purpuratus. These clones can both be identified as MMPs based on the presence of conserved domains such as the cysteine switch, zinc-binding, and hemopexin domains. In addition, both of these genes contain consensus furin cleavage sites and putative transmembrane domains, classifying them as membrane-type MMPs. We have named these clones SpMMP14 and SpMMP16 based on the vertebrate MMPs with which they share the greatest similarity. SpMMP14 is expressed in all cells from the egg to mesenchyme blastula stage embryo. Expression of this gene is strongest in the animal and vegetal poles early in gastrulation and in the animal pole only later in gastrulation. SpMMP16 is expressed at low levels in eggs. Expression of SpMMP16 becomes more pronounced in the vegetal pole region at the blastula and mesenchyme blastula stages and becomes confined to vegetal pole descendants, such as pigment cells, later in development. In the future, we hope to learn more about the possible functions of these genes in sea urchin development.     

The origin of spicule-forming cells in a 'primitive' sea urchin (Eucidaris tribuloides) which appears to lack primary mesenchyme cells

The calcareous larval skeleton of euechinoid sea urchins is synthesized by primary mesenchyme cells which ingress prior to gastrulation. In embryos of the cidaroid sea urchin Eucidaris tribuloides, no mesenchyme cells ingress before gastrulation, yet larvae later contain skeletons. This apparent paradox is resolved by immunochemical, cell lineage and morphological evidence showing that spicule-forming cells of Eucidaris are homologous to primary mesenchyme cells of euechinoids. In particular, these two cell types share expression of two cell lineage-specific gene products, are derived from the same cellular precursors, the micromeres, and undergo a similar migratory phase prior to skeletogenesis. Despite these similarities, there are far fewer spicule-forming cells in Eucidaris than in typical euechinoids and they assume a different pattern during spiculogenesis. The homology between Eucidaris spicule-forming cells and euechinoid primary mesenchyme cells indicates that a heterochrony in the time of spicule-forming cell ingression has occurred since the divergence of their respective lineages.     

Characterization of the upstream region that regulates the transcription of the gene for the precursor to EGF-related peptides,exogastrula-inducing peptides,of the sea urchin Anthocidaris crassispina

The EGIP gene for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are structurally related to the epidermal growth factor, is activated at the onset of gastrulation in subdomains of the embryonic ectoderm. We showed in our previous study that the spatial and temporal regulation of EGIP is conducted by the upstream region from -372 to +194, and that there is an enhancer element between -372 and -210. In this study, we introduced into sea urchin embryos PCR-amplified DNA containing differently truncated EGIP flanking region that was ligated to the GFP reporter gene, and examined the transient expression of the reporter gene, showing that both the -270/-238 and -249/-210 regions were essential for the enhancer activity. We further showed that there is another activating element between -65 and -21, and that even the region between -65 and +194 is sufficient for ectoderm-specific expression of the EGIP gene. The electrophoretic mobility shift assay showed that the -270/-210 enhancer region and the proximal -61/+30 region include specific binding sites for nuclear proteins of sea urchin embryos. Besides these unique sites, the presence of multiple binding sites for GCF1-like nuclear proteins have been revealed in the upstream DNA.