Dissociation of expression patterns of homeodomain transcription factors in the evolution of developmental mode in the sea urchins Heliocidaris tuberculata and H. erythrogramma

The direct-developing sea urchin species Heliocidaris erythrogramma has a radically modified ontogeny. Along with gains of novel features, its entire ectoderm has been reorganized, resulting in the apparent absence of a differentiated oral ectoderm, a major module present in the pluteus of indirect-developing species, such as H. tuberculata. The restoration of an obvious oral ectoderm in H. erythrogrammaxH. tuberculata hybrids, indicates the action of dominant regulatory factors from the H. tuberculata genome. We sought candidate regulatory genes based on the prediction that they should include genes that govern development of the oral ectoderm in the pluteus, but play different roles in H. erythrogramma. Such genes may have a large effect in the evolution of development. Goosecoid (Gsc), Msx, and the sea urchin Abd-B-like gene (Hox11/13b) are present and expressed in both species and the hybrid embryos. Both Gsc and Msx are oral ectoderm specific in H. tuberculata, and show novel and distinct expression patterns in H. erythrogramma. Gsc assumes a novel ectodermal pattern and Msx shifts to a novel and largely mesodermal pattern. Both Gsc and Msx show a restoration of oral ectoderm expression in hybrids. Hox11/13b is not expressed in oral ectoderm in H. tuberculata, but is conserved in posterior spatial expression among H. tuberculata, H. erythrogramma and hybrids, serving as a control. Competitive RT-PCR shows that Gsc, Msx, and Hox11/13b are under different quantitative and temporal controls in the Heliocidaris species and the hybrids. The implications for the involvement of these genes in the rapid evolution of a direct developing larva are discussed.     

Evolutionary modification of T-brain (tbr) expression patterns in sand dollar

The sand dollars are a group of irregular echinoids that diverged from other regular sea urchins approximately 200 million years ago. We isolated two orthologs of T-brain (tbr), Smtbr and Pjtbr, from the indirect developing sand dollar Scaphechinus mirabilis and the direct developing sand dollar Peronella japonica, respectively. The expression patterns of Smtbr and Pjtbr during early development were examined by whole mount in situ hybridization. The expression of Smtbr was first detected in micromere descendants in early blastula stage, similar to tbr expression in regular sea urchins. However, unlike in regular sea urchin, Smtbr expression in middle blastula stage was detected in micromere-descendent cells and a subset of macromere-descendant cells. At gastrula stage, expression of Smtbr was detected in part of the archenteron as well as primary mesenchyme cells. A similar pattern of tbr expression was observed in early Peronella embryos. A comparison of tbr expression patterns between sand dollars and other echinoderm species suggested that broader expression in the endomesoderm is an ancestral character of echinoderms. In addition to the endomesoderm, Pjtbr expression was detected in the apical organ, the animal-most part of the ectoderm.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

Gene expression patterns in a novel animal appendage: the sea urchin pluteus arm

The larval arms of echinoid plutei are used for locomotion and feeding. They are composed of internal calcite skeletal rods covered by an ectoderm layer bearing a ciliary band. Skeletogenesis includes an autonomous molecular differentiation program in primary mesenchyme cells (PMCs), initiated when PMCs leave the vegetal plate for the blastocoel, and a patterning of the differentiated skeletal units that requires molecular cues from the overlaying ectoderm. The arms represent a larval feature that arose in the echinoid lineage during the Paleozoic and offers a subject for the study of gene co-option in the evolution of novel larval features. We isolated new molecular markers in two closely related but differently developing species, Heliocidaris tuberculata and Heliocidaris erythrogramma. We report the expression of a larval arm-associated ectoderm gene tetraspanin, as well as two new PMC markers, advillin and carbonic anhydrase. Tetraspanin localizes to the animal half of blastula stage H. tuberculata and then undergoes a restriction into the putative oral ectoderm and future location of the postoral arms, where it continues to be expressed at the leading edge of both the postoral and anterolateral arms. In H. erythrogramma, its expression initiates in the animal half of blastulae and expands over the entire ectoderm from gastrulation onward. Advillin and carbonic anhydrase are upregulated in the PMCs postgastrulation and localized to the leading edge of the growing larval arms of H. tuberculata but do not exhibit coordinated expression in H. erythrogramma larvae. The tight spatiotemporal regulation of these genes in H. tuberculata along with other ontogenetic and phylogenetic evidence suggest that pluteus arms are novel larval organs, distinguishable from the processes of skeletogenesis per se. The dissociation of expression control in H. erythrogramma suggest that coordinate gene expression in H. tuberculata evolved as part of the evolution of pluteus arms, and is not required for larval or adult development.     

Expression of an Otx gene in the adult rudiment and the developing central nervous system in the vestibula larva of the sea urchin Holopneustes purpurescens

Expression of the Otx gene, HprOtx, from the sea urchin Holopneustes purpurescens, is described during the development of the adult echinoid rudiment in the vestibula larva of this species. The adult rudiment forms directly after gastrulation in the vestibula larva since, unlike the pluteus larva of most other sea urchin species, it is not a feeding larva. The expression is described during the period from hatching to a late vestibula larva. At hatching, HprOtx is expressed throughout the ectoderm of the gastrula. A short time later, expression is absent from the ectoderm on the oral side of the gastrula where the vestibule will form. In an early vestibula larva, HprOtx is not expressed in the ectodermal floor of the vestibule but is expressed in an asymmetric pattern in the aboral ectoderm. As the vestibule invaginates, HprOtx is newly expressed in the ectodermal floor of the vestibule as it develops into the neuroectoderm that is the anlage of the circum-oral central nervous system. The expression is at first in the central part of the floor, then it extends outwards to the ectoderm around the five primary podia and to the epineural folds between the podia. The epineural folds later close to form the radial nerves and the circum-oral nerve ring. In a late vestibula larva, HprOtx is expressed in the radial nerves and the nerve ring. The expression of an Otx gene in the developing echinoid central nervous system is interpreted as an instance of conserved gene expression in echinoderm 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.     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

Patterning mechanisms in the evolution of derived developmental life histories: the role of Wnt signaling in axis formation of the direct-developing sea urchin<Emphasis Type="Italic"> Heliocidaris erythrogramma</Emphasis>

A number of echinoderm species have replaced indirect development with highly modified direct-developmental modes, and provide models for the study of the evolution of early embryonic development. These divergent early ontogenies may differ significantly in life history, oogenesis, cleavage pattern, cell lineage, and timing of cell fate specification compared with those of indirect-developing species. No direct-developing echinoderm species has been studied at the level of molecular specification of embryonic axes. Here we report the first functional analysis of Wnt pathway components in Heliocidaris erythrogramma, a direct-developing sea urchin. We show by misexpression and dominant negative knockout construct expression that Wnt8 and TCF are functionally conserved in the generation of the primary (animal/vegetal) axis in two independently evolved direct-developing sea urchins. Thus, Wnt pathway signaling is an overall deeply conserved mechanism for axis formation that transcends radical changes to early developmental ontogenies. However, the timing of expression and linkages between Wnt8, TCF, and components of the PMC-specification pathway have changed. These changes correlate with the transition from an indirect- to a direct-developing larval life history.Edited by D. Tautz     

Evolutionary convergence in<Emphasis Type="Italic"> Otx</Emphasis> expression in the pentameral adult rudiment in direct-developing sea urchins

Convergence is a significant evolutionary phenomenon. Arrival at similar morphologies from different starting points indicates a strong role for natural selection in shaping morphological phenotypes. There is no evidence yet of convergence in the developmental mechanisms that underlie the evolution of convergent developmental phenotypes. Here we report the expression domains in sea urchins of two important developmental regulatory genes ( Orthodenticle and Runt), and show evidence of molecular convergence in the evolution of direct-developing sea urchins. Indirect development is ancestral in sea urchins. Evolutionary loss of the feeding pluteus stage and precocious formation of the radially symmetric juvenile has evolved independently in numerous sea urchin lineages, thus direct development is an evolutionary convergence. Indirect-developing species do not express Otx during the formation of their five primordial tube feet, the ancestral condition. However, each direct-developing urchin examined does express Otx in the tube feet. Otx expression in the radial arms of direct-developing sea urchins is thus convergent, and may indicate a specific need for Otx use in direct development, a constraint that would make direct development less able to evolve than if there were multiple molecular means for it to evolve. In contrast, Runt is expressed in tube feet in both direct- and indirect-developing species. Because echinoderms are closely related to chordates and postdate the protostome/deuterostome divergence, they must have evolved from bilaterally symmetrical ancestors. Arthropods and chordates use Otx in patterning their anterior axis, and Runt has multiple roles including embryonic patterning in arthropods, and blood and bone cell differentiation in vertebrates. Runt has apparently been co-opted in echinoderms for patterning of pentamery, and Otx in pentameral patterning among direct-developing echinoids. The surprisingly dynamic nature of Otx evolution reinvigorates debate on the role of natural selection vs shared ancestry in the evolution of novel features.     

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.     

Novel population of embryonic secondary mesenchyme cells in the keyhole sand dollar Astriclypeus manni

We have found a novel embryonic cell population in the keyhole sand dollar Astriclypeus manni, which we refer to as lucent fluorescent cells (LFCs). Live LFCs are transparent, but emit autofluorescence after formaldehyde fixation. LFCs become noticeable in the vegetal plate of early gastrulae immediately after the appearance of pigment cells. As development progresses, LFCs increase in number and migrate from the vegetal plate toward the animal pole in a manner similar to pigment cells. Notably, LFCs also migrate into the oral ectoderm, while pigment cells do not. In addition, we determined that there were nearly 300 LFCs per embryo, which greatly exceeds the number of pigment cells. Treatment with the Notch signaling inhibitor N‐[(3,5‐Difluorophenyl)acetyl]‐l ‐alanyl‐2‐phenyl]glycine‐1,1‐dimethylethyl ester (DAPT) resulted in a marked decrease in pigment cell number, but only a modest decrease in LFCs. In DAPT‐treated embryos, LFCs had a distribution pattern similar to pigment cells and were excluded from the oral ectoderm. Unlike other sea urchins, Nodal signaling was not involved in the specification of pigment cells and LFCs in these embryos. Pulse treatment and measurement of cell diameters revealed that LFCs underwent 13–15 cycles of cell division and were specified during the 11th cleavage, one cell cycle later than observed for pigment cells. At the pluteus stage, a cluster of LFCs was observed in the animal plate in addition to two rows of LFCs running along the ciliary band. In addition, dozens of LFCs aligned at the uppermost level of the stomodaeum. Therefore, though the two cell populations share some features, LFCs are considerably different from pigment cells.     

Subequatorial cytoplasm plays an important role in ectoderm patterning in the sea urchin embryo

To gain information on the process of ectoderm patterning, the animal halves of sea urchin embryos were isolated at various stages, and their morphology was examined when control embryos developed into pluteus larvae. The animal halves separated at the 8-cell stage developed into 'dauerblastula', without showing any conspicuous ectoderm differentiation. In contrast, some of the animal halves isolated at the 60-cell stage (after the sixth cleavage) formed a ciliated band and oral opening, suggesting that some patterning signal was transmitted from the vegetal to animal hemisphere during early cleavage. Further patterning of the animal hemisphere did not seem to occur until hatching, since both the animal halves isolated at the 60-cell stage and hatching stage showed the same degree of ectoderm patterning. After hatching, the later animal halves were isolated, the more patterned ectoderm they formed. The animal halves isolated just prior to gastrulation differentiated well-patterned ectoderm. It is of note, however, that the level of separation was a more crucial factor than the timing of separation; even the animal fragments of newly hatched embryos differentiated well-patterned ectoderm if they had been separated at a subequatorial level. This suggests that the signal for ectoderm patterning is transmitted over the equator after hatching, and once the cells in the supra-equatorial region receive the signal, they, in turn, can transmit the signal upwardly. Interestingly, if the third cleavage plane was shifted toward the vegetal pole, the isolated animal pole-side fragments developed into 'embryoids' with fully patterned ectoderm. These results indicate that not the micromere descendants but the subequatorial cytoplasm plays an important role in ectoderm patterning.     

Evolutionary modification of mesenchyme cells in sand dollars in the transition from indirect to direct development

Peronella japonica, an intermediate type of direct-developing sand dollar, forms an abbreviated pluteus, followed by metamorphosis within 3 days without feeding. In this species, ingression of mesenchyme cells starts before hatching and continues until gastrulation, but no typical secondary mesenchyme cells (SMCs) migrate from the tip of the archenteron. Here, I investigated the cell lineage of mesenchyme cells through metamorphosis in P. japonica and found that mesenchyme cells migrating before hatching (early mesenchyme cells [EMCs]) were exclusively derived from micromeres and became larval skeletogenic cells, whereas cells migrating after hatching (late mesenchyme cells [LMCs]) appeared to contain several nonskeletogenic cells. Thus, it is likely that EMCs are homologous to primary mesenchyme cells (PMCs) and LMCs are similar to the SMCs of typical indirect developers, suggesting that heterochrony in the timing of mesenchyme cell ingression may have occurred in this species. EMCs disappeared after metamorphosis and LMCs were involved in adult skeletogenesis. Embryos from which micromeres were removed at the 16-cell stage formed armless plutei that went on to form adult skeletons and resulted in juveniles with normal morphology. These results suggest that in P. japonica, LMCs form adult skeletal elements, whereas EMCs are specialized for larval spicule formation. The occurrence of evolutionary modifications in mesenchyme cells in the transition from indirect to direct development of sand dollars is discussed.     

Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 μM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans. Received: 2 March 1999 / Accepted: 28 June 1999