Novel origins of lineage founder cells in the direct-developing sea urchin Heliocidaris erythrogramma

The lineage and fate of each blastomere in the 32-cell embryo of the direct-developing sea urchin Heliocidaris erythrogramma have been traced by microinjection of tetramethylrhodamine-dextran. The results reveal substantive evolutionary modifications of the ancestral cell lineage pattern of indirect sea urchin development. Significant among these modifications are changes in the time and order of cell lineage segregation: vegetal ectodermal founder cells consistently arise earlier than during indirect development, while internal founder cells generally segregate later and in a different sequence. Modifications have also arisen in proportions of the embryo fated to become various cell types and larval structures. Ectodermal fates, particularly vestibular ectoderm, comprise a greater proportion of the total cellular volume in H. erythrogramma. Among internal cell types, coelom consumes more and endoderm less of the remaining cellular volume than during indirect sea urchin development. Evolutionary modifications are also apparent in the positional origin of larval cell types and structures in H. erythrogramma. These include an apparent tilt in the axis of prospective cell fate relative to the animal-vegetal axis as defined by cleavage planes. Together these evolutionary changes in the cell lineage of H. erythrogramma produce an accelerated loss of dorsoventral symmetry in cell fate relative to indirect development. The extent and diversity of rearrangements in its cell lineage indicate that the non-feeding larva of H. erythrogramma is a highly modified, novel form rather than a degenerate pluteus larva. These same modifications underscore the evolutionarily flexible relationship between cell lineage, gene expression, and larval morphology in sea urchin development.     

Modularity and dissociation in the evolution of gene expression territories in development

SUMMARY Modularity is a salient feature of development and crucial to its evolution. This paper extends modularity to include the concept of gene expression territory, as established for sea urchin embryos. Territories provide a mechanism for partitioning of the cells of a rapidly developing embryo into functional units of a feeding larva. Territories exhibit the characteristics of modules. The paper asks if the embryo and the nonfeeding larva of the direct-developing sea urchin Heliocidaris erythrogramma are organized into gene expression territories, and if its territories correspond to the canonical territories of the pluteus. An analysis of cell lineage and gene expression data for H. erythrogramma shows that skeletogenic cell, coelomic, and vegetal plate gene expression territories are conserved, although they arise from cell lineages distinct from those of the pluteus, and the overall morphology of the larva differs from that of a pluteus. The ectoderm, as in indirect developers, is divided into territories. However, the oral ectodermal territory characteristic of the pluteus is absent in H. erythrogramma. Oral ectoderm is restored in hybrids of H. erythrogramma eggs fertilized by Heliocidaris tuberculata sperm. This indicates that embryonic modules evolve by changes in expression of dominant regulatory genes within territories and that entire modules can be eliminated in evolution of embryos.     

Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos.

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.     

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.     

Oral—aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral–aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral–aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oralaboral axis during sea urchin development.     

Comparative Study of Regulatory Circuits in Two Sea Urchin Species Reveals Tight Control of Timing and High Conservation of Expression Dynamics

Accurate temporal control of gene expression is essential for normal development and must be robust to natural genetic and environmental variation. Studying gene expression variation within and between related species can delineate the level of expression variability that development can tolerate. Here we exploit the comprehensive model of sea urchin gene regulatory networks and generate high-density expression profiles of key regulatory genes of the Mediterranean sea urchin, Paracentrotus lividus (Pl). The high resolution of our studies reveals highly reproducible gene initiation times that have lower variation than those of maximal mRNA levels between different individuals of the same species. This observation supports a threshold behavior of gene activation that is less sensitive to input concentrations. We then compare Mediterranean sea urchin gene expression profiles to those of its Pacific Ocean relative, Strongylocentrotus purpuratus (Sp). These species shared a common ancestor about 40 million years ago and show highly similar embryonic morphologies. Our comparative analyses of five regulatory circuits operating in different embryonic territories reveal a high conservation of the temporal order of gene activation but also some cases of divergence. A linear ratio of 1.3-fold between gene initiation times in Pl and Sp is partially explained by scaling of the developmental rates with temperature. Scaling the developmental rates according to the estimated Sp-Pl ratio and normalizing the expression levels reveals a striking conservation of relative dynamics of gene expression between the species. Overall, our findings demonstrate the ability of biological developmental systems to tightly control the timing of gene activation and relative dynamics and overcome expression noise induced by genetic variation and growth conditions.     

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral–aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral–aboral gene regulatory network. A model for oral–aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral–aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.     

Segregation of oral from aboral ectoderm precursors is completed at fifth cleavage in the embryogenesis of Strongylocentrotus purpuratus

A specific set of founder cells uniquely gives rise to the oral and aboral ectoderms in the regularly developing sea urchin Strongylocentrotus purpuratus. We showed earlier that the polar No and Na (animal oral and animal aboral) blastomeres are specified by third cleavage, while the respective oral and aboral lineage contributions of the left and right NL (animal lateral) blastomeres have not yet segregated from one another at third cleavage. Here we demonstrate by iontophoretic injection of lysyl rhodamine dextran lineage tracer that segregation of oral vs aboral cell fates in the lineages of the NL blastomeres has still not occurred by fourth cleavage, but at fifth cleavage there arise from the NL sublineages founder cells whose progeny contribute exclusively to the aboral ectoderm. The sister cells of these fifth cleavage blastomeres are founder cells that contribute exclusively to oral structures. The aboral ectoderm tracts to which NL derivatives give rise occupy lateral regions of the anterior aboral ectoderm, while the oral structures deriving from the NL blastomeres are the lateral sectors of the ciliated bands. The cells of the ciliated bands do not express aboral ectoderm markers and are considered to constitute the border of the oral region. With these new findings we complete our knowledge of the origins, identities, and fates of the 11 founder cells, the progeny of which exclusively give rise to the aboral ectoderm, and of the 5 founder cells, the progeny of which exclusively produce the oral ectoderm and its derivatives.     

Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.     

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

Direct-developing sea urchins and the evolutionary reorganization of early development.

The evolution of development can be made accessible to study by exploiting closely related species that exhibit distinct ontogenies. The direct-developing sea urchin Heliocidaris erythrogramma is closely related to indirect-developing sea urchins that develop via a feeding larval stage. Superficial consideration would suggest that simple heterochronies resulting in loss of larval features and acceleration of adult features could explain the substitution of direct for indirect development. However, our experiments show that early development has in fact been extensively remodeled, with modified localization of maternal determinants coupled with dissociation of cell cleavage from axis formation resulting in novel patterns of cell lineage differentiation and fate map. Gene expression has undergone concomitant changes.     

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos

In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non-overlapping territories, and overexpression of either one inhibits territory-specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.