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.     

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.     

The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins

The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.     

Evolutionary Conservation of the Larval Serotonergic Nervous System in a Direct Developing Sea Urchin

Development of the larval serotonergic nervous system is examined by indirect immunofluorescence in two congeneric species of sea urchins that exhibit divergent embryonic and larval development. Heliocidar is tuberculata undergoes indirect planktotrophic development via a pluteus larva, whereas Heliocidaris erythrogramma develops directly, passing through a brief, highly derived lecithotrophic larval stage. We have cleared the opaque embryos of H. erythrogramma and discuss internal features of its development. The serotonergic nervous system of H. tuberculata arises in the apical plate at the end of gastrulation and develops into a bilaterally symmetric ganglion lying between the anterolateral arms in the preoral hood. Putatively homologous neurons appear at the apical end of the modified larva of H. erythrogramma well after the completion of gastrulation, coincident with development of the primary podia of the adult rudiment. The neurons form a bilaterally symmetric ganglion whose orientation relative to the vestibule is conserved with respect to that found in planktotrophic larvae. This allows us to define a left and right side for this larva which lacks external points of asymmetry such as a larval mouth. The alteration in the time of nervous system development in H. erythrogramma relative to that of H. tuberculata , and other indirect developers, implicates heterochronies in cellular differentiation as an important component of the evolution of direct development.     

Nodal expression and heterochrony in the evolution of dorsal-ventral and left-right axes formation in the direct-developing sea urchin Heliocidaris erythrogramma

To understand the role of body axes in the evolution of larval form, we use the two sea urchins in the genus Heliocidaris, which have distinctly different larval morphologies. Heliocidaris tuberculata is an indirect-developing sea urchin, which forms a pluteus larva, whereas its sister species, Heliocidaris erythrogramma, exhibits direct development and forms a nonfeeding, ovoid larva. Changes along all three larval axes underlie the differences in larval form associated with each developmental mode. Nodal signaling has recently been implicated as important in establishing the dorsal-ventral (D-V) and left-right (L-R) axes in the indirect-developing sea urchin Paracentrotus lividus. However, because of changes in morphology and timing of morphogenetic events associated with the D-V and L-R axes, respectively, in H. erythrogramma, it was unclear whether nodal played the same roles during direct development. We show that the expression patterns and functions of nodal during H. erythrogramma development are similar to its roles in indirect-developing sea urchins in both D-V and L-R axes formation. However, there are profound changes in gene expression downstream of nodal signaling along the D-V axis and major heterochronies in the execution of the function of nodal along the L-R axis. These highly modified events are linked to the dramatic modifications of larval morphology that have occurred during the evolution of direct development in H. erythrogramma.     

Co-option and dissociation in larval origins and evolution: the sea urchin larval gut

SUMMARY The origin of marine invertebrate larvae has been an area of controversy in developmental evolution for over a century. Here, we address the question of whether a pelagic "larval" or benthic "adult" morphology originated first in metazoan lineages by testing the hypothesis that particular gene co-option patterns will be associated with the origin of feeding, indirect developing larval forms. Empirical evidence bearing on this hypothesis is derivable from gene expression studies of the sea urchin larval gut of two closely related but differently developing congenerics, Heliocidaris tuberculata (feeding indirect-developing larva) and H. erythrogramma (nonfeeding direct developer), given two subsidiary hypotheses. (1) If larval gut gene expression in H. tuberculata was co-opted from an ancestral adult expression pattern, then the gut expression pattern will remain in adult H. erythrogramma despite its direct development. (2) Genes expressed in the larval gut of H. tuberculata will not have a coordinated expression pattern in H. erythrogramma larvae due to loss of a functional gut. Five structural genes expressed in the invaginating archenteron of H. tuberculata during gastrulation exhibit substantially different expression patterns in H. erythrogramma with only one remaining endoderm specific. Expression of these genes in the adult of H. erythrogramma and larval gut of H. tuberculata , but not in H. erythrogramma larval endoderm, supports the hypothesis that they first played roles in the formation of adult structures and were subsequently recruited into larval ontogeny during the origin and evolution of feeding planktotrophic deuterostome larvae.     

Ciliated band structure in planktotrophic and lecithotrophic larvae of Heliocidaris species (Echinodermata: Echinoidea): a demonstration of conservation and change

The evolution of lecithotrophic (non-feeding) development in sea urchins is associated with reduction or loss of structures found in the planktotrophic (feeding) echinopluteus larvae. Reductions or losses of larval feeding structures include pluteal arms, their supporting skeleton and the ciliated band that borders them. The barrel-shaped lecithotrophic larva of Heliocidaris erythrogramma has, at its posterior end, two or three ciliated band segments comprised of densely packed, elongate cilia. These cilia may be expressions of the epaulettes that would have been present in an ancestral larval form, represented today by the feeding echinopluteus of H. tuberculata . We compared the development and cellular organization of the larval ciliary structures of both Heliocidaris species to assess whether the ciliary bands of H. erythrogramma are expressions of the feeding ciliated band or epaulettes of an echinopluteus. Epaulette development in feeding larvae of H. tuberculata involves separation of specific parts of the ciliated band from the rest of the feeding ciliated band, hyperplastic addition of ciliated cells and hypertrophic growth of the cilia. Like epaulettes, the ciliated bands of H. erythrogramma are composed of long spindle-shaped cells arranged in a cup-shaped collection that bulges into the blastocoel; and these cells have elongated cilia. In their developmental origin and topological arrangement however, the ciliated bands of H. erythrogramma correspond more closely with parts of the pluteal feeding ciliated band than with epaulettes. The larvae of this echinoid appear to develop epaulette-like bands from parts of the original (but reduced) feeding ciliated band. The evolution of development in H. erythrogramma has thus involved both conservation and change in echinopluteal ciliary structures.     

Evolutionary Modification of Echinoid Sperm Correlates with Developmental Mode

A significant fraction of living sea urchin species have completely or partially eliminated the pluteus larval stage and instead develop directly from embryo to adult. Direct developing sea urchins develop from large buoyant eggs. We present data to show that evolution of these large eggs is accompanied by the evolution of spermatozoa with elogate heads, in contrast with the conical sperm heads typical of most echinoids. Two congeneric Australian species, Heliocidaris tuberculata , which develops via a pluteus, and H. erythogramma , a direct developer, were investigated in detail. The sperm of H. erythrogramma have an elongate head (11 μm in length) as compared to the conical sperm head (5.6 μm) of H. tuberculata . Electrophoretic analysis of the sperm histones indicates that no unusual histones or protamines are associated with modified head morphology. Genome sizes were determined by flow cytometry. H. erythrogramma has a haploid genome size of 1.3 pg as compared to a haploid genome size of 0.95 pg for H. tuberculata . Other direct developing echinoids have elongate sperm heads, and co-evolution of gametes is indicated as a common feature of evolution of direct development in echinoids. The most extreme case, the direct developing cidaroid sea urchin, Phyllacanthus parvispinus , possesses the longest and narrowest sperm head (20 μm × 1 μm) ever observed in an echinoid.     

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.     

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.     

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.     

A novel ontogenetic pathway in hybrid embryos between species with different modes of development

To investigate the bases for evolutionary changes in developmental mode, we fertilized eggs of a direct-developing sea urchin, Heliocidaris erythrogramma, with sperm from a closely related species, H. tuberculata, that undergoes indirect development via a feeding larva. The resulting hybrids completed development to form juvenile adult sea urchins. Hybrids exhibited restoration of feeding larval structures and paternal gene expression that have been lost in the evolution of the direct-developing maternal species. However, the developmental outcome of the hybrids was not a simple reversion to the paternal pluteus larval form. An unexpected result was that the ontogeny of the hybrids was distinct from either parental species. Early hybrid larvae exhibited a novel morphology similar to that of the dipleurula-type larva typical of other classes of echinoderms and considered to represent the ancestral echinoderm larval form. In the hybrid developmental program, therefore, both recent and ancient ancestral features were restored. That is, the hybrids exhibited features of the pluteus larval form that is present in both the paternal species and in the immediate common ancestor of the two species, but they also exhibited general developmental features of very distantly related echinoderms. Thus in the hybrids, the interaction of two genomes that normally encode two disparate developmental modes produces a novel but harmonious ontongeny.     

Co-option of an oral-aboral patterning mechanism to control left-right differentiation: the direct-developing sea urchin Heliocidaris erythrogramma is sinistralized, not ventralized, by NiCl2

Larval dorsoventral (DV) and left-right (LR) axial patterning unfold progressively in sea urchin development, leading to commitment of the major embryonic regions by the gastrula stage. The direct-developing sea urchin Heliocidaris erythrogramma has lost oral-aboral differentiation along the DV axis but has accelerated vestibular ectoderm development on the left side. NiCl(2) radializes indirect-developing sea urchins by shifting cells toward a ventral fate (oral ectoderm). We treated embryos of H. erythrogramma and the indirect-developing H. tuberculata with NiCl(2). H. tuberculata was ventralized exactly like other indirect developers, establishing that basic patterning mechanisms are conserved in this genus. H. erythrogramma was also radialized; timing, dosage response, and some morphological features were similar to those in other sea urchins. Ectodermal explant and recombination experiments demonstrate that the effect of nickel is autonomous to the ectoderm, another feature in common with indirect developers. However, H. erythrogramma is distinctly sinistralized rather than ventralized, its cells shifting toward a left-side fate (vestibular ectoderm). This geometric contrast in the midst of pervasive functional similarity suggests that nickel-sensitive processes in H. erythrogramma axial patterning, homologous to those in indirect developers, have been redeployed, and hence co-opted, from their ancestral role in DV axis determination to a new role in LR axis determination. We discuss DV and LR axial patterning and their evolutionary transformation.     

Single-copy DNA distance between two congeneric sea urchin species exhibiting radically different modes of development

We have investigated the differences between nuclear genomes of two purportedly congeneric species of sea urchin that differ radically in early development. Heliocidaris tuberculata develops by means of a typical pluteus larva, whereas H. erythrogramma develops directly from an egg that is 100-fold the volume of the H. tuberculata egg. Reassociation kinetic analysis shows that the kinetic components of the genomic DNA from the two species are essentially the same. No single repeat component explains the 30% difference between the H. erythrogramma and H. tuberculata genomes. Reciprocal hybridization of tracer-labeled single-copy DNA fractions between these species indicates that approximately 50% of the single-copy DNA is sufficiently similar to form hybrids at standard hybridization criterion. Thermal denaturation profiles of the hybridized single-copy DNA sequence yields median (T50H) values of 13.8 degrees-16.5 degrees C. This result suggests a divergence time of 10-13 Mya, which is comparable to divergence times between congeneric sea urchin species in other genera that do not differ significantly in development. Radical differences in early developmental processes can evolve rapidly between closely related forms.     

Novel gene expression patterns in hybrid embryos between species with different modes of development

SUMMARY Cross-species hybrids between eggs of the direct-developing sea urchin, Heliocidaris erythrogramma , and sperm from its congeneric indirect-developing species, Heliocidaris tuberculata, show restoration of features of the paternal feeding pluteus larva, including the gut, and pluteus spicular skeleton. Unlike other reported sea urchin cross-species hybrids, Heliocidaris hybrids express genes derived from both maternal and paternal species at high levels. Ectodermal cell types, which differ radically between the two parental species, are of intermediate form in the hybrids. Gene expression patterns in hybrid embryo tissues represent a number of combinations of parental gene expression patterns: genes that are not expressed in one paternal species, but are expressed in hybrids as in the expressing parent; genes that show additive expression patterns plus novel sites of expression; a gene that is misexpressed in the hybrids; and genes expressed identically in both parents and in hybrids. The results indicate that both conserved and novel gene regulatory interactions are present. Only one gene, CyIII actin , has lost cell-type-specific regulation in the hybrids. Hybrids thus reveal that disparate parental genomes, each with its own genic regulatory system, can produce in combination a novel gene expression entity with a unique ontogeny. This outcome may derive from conserved gene regulatory regions in downstream genes of both parental species responding in conserved ways to higher-level regulators that determine modular gene expression territories.     

Adaptive evolution of bindin in the genus Heliocidaris is correlated with the shift to direct development

Abstract Sea urchins are widely used to study both fertilization and development. In this study we combine the two fields to examine the evolution of reproductive isolation in the genus Heliocidaris . Heliocidaris tuberculata develops indirectly via a feeding larva, whereas the only other species in the genus, H. erythrogramma , has evolved direct development through a nonfeeding larva. We estimated the time of divergence between H. erythrogramma and H. tuberculata from mitochondrial DNA divergence, quantified levels of gametic compatibility between the two species in cross-fertilization assays, and examined the mode of evolution of the sperm protein bindin by sequencing multiple alleles of the two species. Bindin is the major component of the sea urchin sperm acrosomal vesicle, and is involved in sperm-egg attachment and fusion. Based on our analyses, we conclude that: the two species of Heliocidaris diverged less than five million years ago, indicating that direct development can evolve rapidly in sea urchins; since their divergence, the two species have become gametically incompatible; Heliocidaris bindin has evolved under positive selection; and this positive selection is concentrated on the branch leading to H. erythrogramma . Three hypotheses can explain the observed pattern of selection on bindin: (1) it is a correlated response to the evolution of direct development in H. erythrogramma; (2) it is the result of an intraspecific process acting in H. erythrogramma but not in H. tuberculata; or (3) it is the product of reinforcement on the species that invests more energy into each egg to avoid hybridization.     

Regulatory punctuated equilibrium and convergence in the evolution of developmental pathways in direct-developing sea urchins

We made hybrid crosses between closely and distantly related sea urchin species to test two hypotheses about the evolution of gene regulatory systems in the evolution of ontogenetic pathways and larval form. The first hypothesis is that gene regulatory systems governing development evolve in a punctuational manner during periods of rapid morphological evolution but are relatively stable over long periods of slow morphological evolution. We compared hybrids between direct and indirect developers from closely and distantly related families. Hybrids between eggs of the direct developer Heliocidaris erythrogramma and sperm of the 4-million year distant species H. tuberculata, an indirect developer, restored feeding larval structures and paternal gene expression that were lost in the evolution of the direct-developing maternal parent. Hybrids resulting from the cross between eggs of H. erythrogramma and sperm of the 40-million year distant indirect-developer Pseudoboletia maculata are strikingly similar to hybrids between the congeneric hybrids. The marked similarities in ontogenetic trajectory and morphological outcome in crosses of involving either closely or distantly related indirect developing species indicates that their regulatory mechanisms interact with those of H. erythrogramma in the same way, supporting remarkable conservation of molecular control pathways among indirect developers. Second, we tested the hypothesis that convergent developmental pathways in independently evolved direct developers reflect convergence of the underlying regulatory systems. Crosses between two independently evolved direct-developing species from two 70-million year distant families, H. erythrogramma and Holopneustes purpurescens, produced harmoniously developing hybrid larvae that maintained the direct mode of development and did not exhibit any obvious restoration of indirect-developing features. These results are consistent with parallel evolution of direct-developing features in these two lineages.