Influence of cell geometry on division-plane positioning.

The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces.

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Light-induced, spatiotemporal control of protein in the developing embryo of the sea urchin.

Differential protein regulation is a critical biological process that regulates cellular activity and controls cell fate determination. It is especially important during early embryogenesis when post-transcriptional events predominate differential fate specification in many organisms. Light-induced approaches have been a powerful technology to interrogate protein functions with temporal and spatial precision, even at subcellular levels within a cell by controlling laser irradiation on the confocal microscope. However, application and efficacy of these tools need to be tested for each model system or for the cell type of interest because of the complex nature of each system. Here, we introduce two types of light-induced approaches to track and control proteins at a subcellular level in the developing embryo of the sea urchin. We found that the photoconvertible fluorescent protein Kaede is highly efficient to distinguish pre-existing and newly synthesized proteins with no apparent phototoxicity, even when interrogating proteins associated with the mitotic spindle. Further, chromophore-assisted light inactivation (CALI) using miniSOG successfully inactivated target proteins of interest in the vegetal cortex and selectively delayed or inhibited asymmetric cell division. Overall, these light-induced manipulations serve as important molecular tools to identify protein function for for subcellular interrogations in developing embryos.

Conserved regulatory state expression controlled by divergent developmental gene regulatory networks in echinoids.

Evolution of the animal body plan is driven by changes in developmental gene regulatory networks (GRNs), but how networks change to control novel developmental phenotypes remains, in most cases, unresolved. Here, we address GRN evolution by comparing the endomesoderm GRN in two echinoid sea urchins, Strongylocentrotus purpuratus and Eucidaris tribuloides, with at least 268 million years of independent evolution. We first analyzed the expression of twelve transcription factors and signaling molecules of the S. purpuratus GRN in E. tribuloides embryos, showing that orthologous regulatory genes are expressed in corresponding endomesodermal cell fates in the two species. However, perturbation of regulatory genes revealed that important regulatory circuits of the S. purpuratus GRN are significantly different in E. tribuloides For example, mesodermal Delta/Notch signaling controls exclusion of alternative cell fates in E. tribuloides but controls mesoderm induction and activation of a positive feedback circuit in S. purpuratus These results indicate that the architecture of the sea urchin endomesoderm GRN evolved by extensive gain and loss of regulatory interactions between a conserved set of regulatory factors that control endomesodermal cell fate specification.

Neurogenesis in sea urchin embryos and the diversity of deuterostome neurogenic mechanisms.

A single origin to the diverse mechanisms of metazoan neurogenesis is suggested by the involvement of common signaling components and similar classes of transcription factors. However, in many forms we lack details of where neurons arise, patterns of cell division, and specific differentiation pathway components. The sea urchin larval nervous system is composed of an apical organ, which develops from neuroepithelium and functions as a central nervous system, and peripheral neurons, which differentiate in the ciliary band and project axons to the apical organ. To reveal developmental mechanisms of neurogenesis in this basal deuterostome, we developed antibodies to SoxC, SoxB2, ELAV and Brn1/2/4 and used neurons that develop at specific locations to establish a timeline for neurogenesis. Neural progenitors express, in turn, SoxB2, SoxC, and Brn1/2/4, before projecting neurites and expressing ELAV and SynB. Using pulse-chase labeling of cells with a thymidine analog to identify cells in S-phase, we establish that neurons identified by location are in their last mitotic cycle at the time of hatching, and S-phase is coincident with expression of SoxC. The number of cells expressing SoxC and differentiating as neurons is reduced in embryos injected with antisense morpholino oligonucleotides to SoxC, SoxB2 or Six3. Injection of RNA encoding SoxC into eggs does not enhance neurogenesis. In addition, inhibition of FGF receptors (SU5402) or a morpholino to FGFR1 reduces expression of SoxC. These data indicate that there are common features of neurogenesis in deuterostomes, and that sea urchins employ developmental mechanisms that are distinct from other ambulacraria.

Single cell RNA-seq in the sea urchin embryo show marked cell-type specificity in the Delta/Notch pathway.

Sea urchin embryos are excellent for in vivo functional studies because of their transparency and tractability in manipulation. They are also favorites for pharmacological approaches since they develop in an aquatic environment and addition of test substances is straightforward. A concern in many pharmacological tests though is the potential for pleiotropic effects that confound the conclusions drawn from the results. Precise cellular interpretations are often not feasible because the impact of the perturbant is not known. Here we use single-cell mRNA (messenger RNA) sequencing as a metric of cell types in the embryo and to determine the selectivity of two commonly used inhibitors, one each for the Wnt and the Delta-Notch pathways, on these nascent cell types. We identified 11 distinct cell types based on mRNA profiling, and that the cell lineages affected by Wnt and Delta/Notch inhibition were distinct from each other. These data support specificity and distinct effects of these signaling pathways in the embryo and illuminate how these conserved pathways selectively regulate cell lineages at a single cell level. Overall, we conclude that single cell RNA-seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories.

Microsphere packages of carotenoids: intact sea urchin eggs tracked by Raman spectroscopy tools.

Although natural exposure to ambient UV radiation in oligotrophic seawater at small depths can reach the levels responsible for cellular damage, the sea urchin Paracentrotus lividus is frequently in such sites, particularly on the southern Adriatic Sea shore. Spawning their eggs and spending their early life stage in rocky shores at depths of 0.5-2 m are the results of their successful adaptation strategies, although adults may dwell at greater depths. Surprisingly, there is a paucity of reports regarding the carotenoid content in sea urchin eggs. Beyond their important role in photoprotection against high UV exposure, cell division and early development, the content and distribution of carotenoids contribute to the successful survival of sea urchins and also determine the color of their gonads (roe), which is of commercial importance as a delicacy. Herein, for the first time, we have described the carotenoid content and distribution in intact, freshly released eggs of P. lividus species, non-destructively employing resonance Raman spectroscopy and imaging; near-infrared Raman spectroscopy revealed additional molecular carotenoid content. Echinenone and ß-carotene resonance Raman signals were the most intense, and they were identified as the principal carotenoids that are preferentially accumulated in eggs rather than in gonads. Raman imaging in confocal mode revealed the uniform distribution of the carotenoid signal over the whole eggs, while the distribution of proteins appeared spotted. Egg carotenoids generally maintained their identity after 2 months of dry storage, with slight signs of C[double bond, length as m-dash]C bond oxidation. The potential utilization of P. lividus sea urchin eggs as valuable microsphere packages of native carotenoids is discussed.

KirrelL, a member of the Ig-domain superfamily of adhesion proteins, is essential for fusion of primary mesenchyme cells in the sea urchin embryo.

In the sea urchin embryo, primary mesenchyme cells (PMCs) adhere to one another and fuse via filopodia, forming cable-like structures within which skeletal rods are deposited. Although this process was first described more than a century ago, molecules that participate in PMC adhesion and fusion have not been identified. Here we show that KirrelL, a PMC-specific, Ig domain-containing transmembrane protein, is essential for PMC fusion, probably by mediating filopodial adhesions that are a pre-requisite for subsequent membrane fusion. We show that KirrelL is not required for PMC specification, migration, or for direct filopodial contacts between PMCs. In the absence of KirrelL, however, filopodial contacts do not result in fusion. kirrelL is a member of a family of closely related, intronless genes that likely arose through an echinoid-specific gene expansion, possibly via retrotransposition. Our findings are significant in that they establish a direct linkage between the transcriptional network deployed in the PMC lineage and an effector molecule required for a critically important PMC morphogenetic process. In addition, our results point to a conserved role for Ig domain-containing adhesion proteins in facilitating cell fusion in both muscle and non-muscle cell lineages during animal development.

TGF-ß sensu stricto signaling regulates skeletal morphogenesis in the sea urchin embryo.

Cell-cell signaling plays a prominent role in the formation of the embryonic skeleton of sea urchins, but the mechanisms are poorly understood. In the present study, we uncover an essential role for TGF-ß sensu stricto signaling in this process. We show that TgfbrtII, a type II receptor dedicated to signaling through TGF-ß sensu stricto, is expressed selectively in skeletogenic primary mesenchyme cells (PMCs) during skeleton formation. Morpholino (MO) knockdowns and studies with a specific TgfbrtII inhibitor (ITD-1) in both S. purpuratus and Lytechinus variegatus embryos show that this receptor is required for biomineral deposition. We provide pharmacological evidence that Alk4/5/7 is the cognate TGF-ß type I receptor that pairs with TgfbrtII and show by inhibitor treatments of isolated micromeres cultured in vitro that both Alk4/5/7 and TgfbrtII function cell-autonomously in PMCs. Gene expression and gene knockdown studies suggest that TGF-ß sensu stricto may be the ligand that interacts with TgfbrtII and support the view that this TGF-ß superfamily ligand provides an essential, permissive cue for skeletogenesis, although it is unlikely to provide spatial patterning information. Taken together, our findings reveal that this model morphogenetic process involves an even more diverse suite of cell signaling pathways than previously appreciated and show that PMCs integrate a complex set of both generalized and spatially localized cues in assembling the endoskeleton.

MAPK/ERK activity is required for the successful progression of mitosis in sea urchin embryos.

Using sea urchin embryos, we demonstrate that the MEK/MAPK/ERK cascade is essential for the proper progression of the cell cycle. Activation of a limited fraction of MAPK/ERK is required between S-phase and M-phase. Neither DNA replication nor CDK1 activation are impacted by the inhibition of this small active MAPK/ERK fraction. Nonetheless, the chromatin and spindle organisations are profoundly altered. Early morphological disorders induced by the absence of MAPK/ERK activation are correlated with an important inhibition of global protein synthesis and modification in the cyclin B accumulation profile. After appearance of morphological disorders, there is an increase in the level of the inhibitor of protein synthesis, 4E-BP, and, ultimately, an activation of the spindle checkpoint. Altogether, our results suggest that MAPK/ERK activity is required for the synthesis of (a) protein(s) implicated in an early step of chromatin /microtubule attachment. If this MAPK/ERK-dependent step is not achieved, the cell activates a new checkpoint mechanism, involving the reappearance of 4E-BP that maintains a low level of protein translation, thus saving cellular energy.

Notch-mediated lateral inhibition is an evolutionarily conserved mechanism patterning the ectoderm in echinoids.

Notch signaling is a crucial cog in early development of euechinoid sea urchins, specifying both non-skeletogenic mesodermal lineages and serotonergic neurons in the apical neuroectoderm. Here, the spatial distributions and function of delta, gcm, and hesc, three genes critical to these processes in euechinoids, are examined in the distantly related cidaroid sea urchin Eucidaris tribuloides. Spatial distribution and experimental perturbation of delta and hesc suggest that the function of Notch signaling in ectodermal patterning in early development of E. tr ibuloides is consistent with canonical lateral inhibition. Delta transcripts were observed in t he archenteron, apical ectoderm, and lateral ectoderm in gastrulating e mbryos of E. tribuloides. Perturbation of Notch signaling by either delta morpholino or treatment of DAPT downregulated hesc and upregulated delta and gcm, resulting in ectopic expression of delta and gcm. Similarly, hesc perturbation mirrored the effects of delta perturbation. Interestingly, perturbation of delta or hesc resulted in more cells expressing gcm and supernumerary pigment cells, suggesting that pigment cell proliferation is regulated by Notch in E. tribuloides. These results are consistent with an evolutionary scenario whereby, in the echinoid ancestor, Notch signaling was deployed in the ectoderm to specify neurogenic progenitors and controlled pigment cell proliferation in the dorsal ectoderm.

Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics.

Starfish and sea urchin are excellent models to study the mechanisms that regulate oocyte maturation and egg activation. Hormonal stimulation of starfish oocytes and their following interaction with spermatozoa induce rapid changes of F-actin and Ca2+ increases which are prerequisites for normal fertilization and development. Fully grown oocytes isolated from the gonads of starfish contain a large nucleus (∼60-70 µm) (termed germinal vesicle, GV), which is arrested at the first prophase of meiosis. If inseminated, these immature oocytes are penetrated by additional spermatozoa. However, starfish oocytes naturally shed into the sea have already initiated the (meiotic) maturation and are normally fertilized between GV breakdown and the extrusion of the first polar body. This is considered the optimum period to ensure monospermic instead of polyspermic fertilization. By contrast, sea urchin eggs are fertilized only after being fully matured, i.e., at the end of the two meiotic divisions. Here, we provide a comparative review of the role of the actin cytoskeleton in oocyte maturation and fertilization in starfish and sea urchin. It has become increasingly evident that the exquisite regulation of the cortical F-actin is involved in nearly all aspects of the molecular events taking place during the progression of meiotic maturation and fertilization.

Genome-wide assessment of differential effector gene use in embryogenesis.

Six different populations of cells were isolated by fluorescence-activated cell sorting from disaggregated late blastula- and gastrula-stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered bacterial artificial chromosomes producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicated the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1-2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus, at the effector gene level, a dramatic, cell type-specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred.

Peanut agglutinin specifically binds to a sperm region between the nucleus and mitochondria in tunicates and sea urchins.

Peanut agglutinin (PNA) is an established marker of the mammalian acrosome. However, we observed that PNA specifically binds to a unique intracellular structure alongside the nucleus in ascidian sperm. Here, we characterize the PNA-binding structure in sperm of marine invertebrates. PNA bound to the region between the mitochondrion and nucleus in spermatozoa of ascidians, sea urchins, and an appendicularian. However, PNA-binding substances were not exposed by the calcium ionophore ionomycin in three ascidian species, indicating that it is a distinct structure from the acrosome. Instead, the ascidian PNA-binding region was shed with the mitochondrion from the sperm head via an ionomycin-induced sperm reaction. The ascidian PNA-binding substance appeared to be solubilized with SDS, but not Triton X-100, describing its detergent resistance. Lectins, PHA-L4 , SSA, and MAL-I were detected at an area similar to the PNA-binding region, suggesting that it contains a variety of glycans. The location and some of the components of the PNA-binding region were similar to known endoplasmic reticulum (ER)-derived structures, although the ER marker concanavalin A accumulated at an area adjacent to but not overlapping the PNA-binding region. Therefore, we conclude that ascidian sperm possess a non-acrosomal, Triton-resistant, glycan-rich intracellular structure that may play a general role in reproduction of tunicates and sea urchins given its presence across a wide taxonomic range.

Cryopreservation: Extending the viability of biological material from sea urchin (Echinometra lucunter) in ecotoxicity tests.

The sea urchin, Echinometra lucunter, is widely used in embryo-larval tests for ecotoxicological studies in Brazil and other countries. For each test, sea urchins are collected from the wild and this can cause impact on wild populations and it is limited by the weather and season which in turn limits the ability to carry out the tests. Cryopreservation is a method of live biological material storage at low temperature and can be used for long periods with little decline in viability, reducing the number of animals taken from the wild and enabling testing to be carried out on demand, irrespective of spawning season or location. In this study, 15 combinations of cryoprotective agents (CPAs) were evaluated on spermatozoa, subjected to a rapid cooling curve followed by immersion in liquid nitrogen. Twenty-four CPA combinations were evaluated on eggs subjected to a more gradual cooling curve in nitrogen vapor down to -35 °C and then plunging in liquid nitrogen. Fertilization tests using cryopreserved spermatozoa gave high pluteus larvae yields (≈80%) when concentrations of 10.5% or 13.65% ME2SO or 13.65% ME2SO+15.75% sucrose were used. The higher concentrations of ME2SO plus sucrose were more effective at maintaining the fertilization capacity of spermatozoa post-thawing. Egg cryopreservation was not successful with 0% fertilization observed post-thawing. The results suggest that it is feasible to implement spermatozoa cryopreservation as technological innovation to create a sperm bank for E. lucunter, which can be used in ecotoxicological tests, bringing benefits for researches and contributing to the conservation of the species.

A sea urchin in vivo model to evaluate Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti-EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti-EMT candidates.

Cilia are required for asymmetric nodal induction in the sea urchin embryo.

BACKGROUND: Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. RESULTS: Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. CONCLUSIONS: Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

Morphological and genetic analyses reveal a cryptic species complex in the echinoid Echinocardium cordatum and rule out a stabilizing selection explanation.

Preliminary analyses revealed the presence of at least five mitochondrial clades within the widespread sea urchin Echinocardium cordatum (Spatangoida). In this study, we analyzed the genetic (two mitochondrial and two nuclear sequence loci) and morphological characteristics (20 indices) from worldwide samples of this taxon to establish the species limits, morphological diversity and differentiation. Co-occurring spatangoid species were also analyzed with mitochondrial DNA. The nuclear sequences confirm that mitochondrial lineages correspond to true genetic entities and reveal that two clades (named A and B1) hybridize in their sympatry area, although a more closely related pair of clades (B1 and B2), whose distributions widely overlap, does not display hybridization. The morphology of all E. cordatum clade pairs was significantly differentiated, but no morphological diagnostic character was evidenced. By contrast, other spatangoid species pairs that diverged more recently than the E. cordatum clades display clear diagnostic characters. Morphological diversity thus appears responsible for the absence of diagnostic characters, ruling out stabilizing selection, a classical explanation for cryptic species. Alternative classical explanations are (i) environmental plasticity or (ii) a high diversity of genes determining morphology, maintained by varying environmental conditions. We suggest a new hypothesis that the observed morphological diversity is selectively neutral and reflects high effective population sizes in the E. cordatum complex. It is supported by the higher abundance of this taxon compared with other taxa, a trend for the genetic and morphological diversity to be correlated in Europe, and the higher genetic and morphological diversities found in clades of E cordatum (except B1) than in other spatangoid samples in Europe. However, the Pacific clades do not confirm these trends.

Roles of hesC and gcm in echinoid larval mesenchyme cell development.

To understand the roles of hesC and gcm during larval mesenchyme specification and differentiation in echinoids, we performed perturbation experiments for these genes in two distantly related euechinoids, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. The number of larval mesenchyme cells increased when the translation of hesC was inhibited, thereby suggesting that hesC has a general role in larval mesenchyme development. We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. Simultaneous inhibition of the translation of hesC and gcm induced a significant increase in the number of skeletogenic cells, which suggests that gcm functions in skeletogenic fate repression. Based on these observations, we suggest that: (i) hesC participates in some general aspects of mesenchymal cell development; and (ii) gcm is involved in the mechanism responsible for the binary specification of skeletogenic and pigment cell fates.

Ancient animal microRNAs and the evolution of tissue identity.

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.