An easy and rapid staining method for confocal microscopic observation and reconstruction of three-dimensional images of echinoderm larvae and juveniles.

The morphologies of the internal organs of echinoderm larvae and juveniles are difficult to study using conventional optical microscopes because of their structural complexity and opaqueness. This paper describes an easy and rapid protocol involving Nile blue staining followed by benzyl alcohol/benzyl benzoate (BABB) clearing to overcome this limitation. This method was developed for a three-dimensional (3D) analysis of the internal structures of advanced larvae and juveniles of echinoderms (the sea lily Metacrinus rotundus, the sea urchin Hemicentrotus pulcherrimus, and the sand dollar Scaphechinus mirabilis) and is suitable for obtaining serial optical images by confocal microscopy without the use of specific antibodies or special reagents for labeling. Nile blue is an easy-to-use stain that offers several advantages for confocal microscopy such as it can stain various tissues with strong fluorescent signals without substantial bleaching during observation. We found that the strong fluorescence signal of Nile blue quickly yielded clear high-resolution optical section images for 3D reconstruction. BABB clearing rendered opaque larvae highly transparent. The clearing procedure was also easy and quick. During the process, agarose embedding prior to staining and clearing was found to be critical for handling the samples of less than 500-µm length and stabilizing their orientations. To conclude, the protocol described is useful for performing a rapid and accurate 3D morphological analysis of echinoderm larvae and juveniles.

pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

Cidaroids, clypeasteroids, and spatangoids: Procurement, culture, and basic methods.

This chapter describes practical methods and key points for using non-camarodont echinoids including cidaroids (Order Cidaroida), clypeasteroids (also known as sand dollars; Order Clypeasteroida), and spatangoids (also known as heart urchins; Order Spatangoida) as experimental subjects for biological studies. The content described here is based on six Japanese species of echinoids (Astriclypeus manni, Clypeaster japonicus, Echinocardium cordatum, Peronella japonica, Prionocidaris baculosa, and Scaphechinus mirabilis). Specific topics addressed in this chapter include the collection and maintenance of adults, embryonic culture, and experimental procedures for micromanipulations, whole mount in situ hybridization, and immunological experiments.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

Comparative studies on the skeletogenic mesenchyme of echinoids.

Skeletogenic mesenchyme cells in echinoids are suitable for studying developmental mechanisms, and have been used extensively. Most of these studies have been performed on species in the order Camarodonta, which are modern echinoids (subclass Euechinoidea) and are considered "model" echinoid species. In contrast, species belonging to other orders are studied less frequently, especially investigations of their molecular developmental biology such as gene regulatory networks. Recent studies on mesenchyme development in non-camarodont species suggest that these species are potential sources of comparative information to elucidate the mechanisms underlying skeletogenic mesenchyme development. In this review, the importance of using comparative data to understand development and evolution is discussed.

Characterization of paramyosin and thin filaments in the smooth muscle of acorn worm, a member of hemichordates.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.

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.

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

Expession patterns of mesenchyme specification genes in two distantly related echinoids, Glyptocidaris crenularis and Echinocardium cordatum.

The molecular mechanism of the larval mesenchyme cell specification in echinoids has been well analyzed. However, most of the data have been provided by studies of a single group of echinoids, the order Camarodonta. Little is known about this mechanism in other echinoid orders. We examined the expression patterns of mesenchyme specification genes, micro1, hesC, alx1, tbr, ets1, cyp1, and gcm, in the two non-Camarodonta echinoids, Glyptocidaris crenularis and Echinocardium cordatum. We found that the expression patterns of some genes contained characteristics that were unique to one of the species; others were shared by the two species. Some of the shared characteristics of G. crenularis and E. cordatum are not found in the species belonging to Camarodonta, suggesting the derived status of this order. The expression of ets1 in E. cordatum aboral ectoderm is one of the molecular level modifications possibly related to an evolutionarily novel larval structure, the posterior process. Our results suggest that a considerable number of modifications in the mesenchyme specification mechanisms have been introduced during the echinoid evolution.

Larval mesenchyme cell specification in the primitive echinoid occurs independently of the double-negative gate.

Echinoids (sea urchins) are divided into two major groups - cidaroids (a 'primitive' group) and euechinoids (a 'derived' group). The cidaroids are a promising model species for understanding the ancestral developmental mechanisms in echinoids, but little is known about the molecular mechanisms of cidaroid development. In euechinoids, skeletogenic mesenchyme cell specification is regulated by the double-negative gate (DNG), in which hesC represses the transcription of the downstream mesenchyme specification genes (alx1, tbr and ets1), thereby defining the prospective mesenchyme region. To estimate the ancestral mechanism of larval mesenchyme cell specification in echinoids, the expression patterns and roles of mesenchyme specification genes in the cidaroid Prionocidaris baculosa were examined. The present study reveals that the expression pattern and function of hesC in P. baculosa were inconsistent with the DNG model, suggesting that the euechinoid-type DNG is not utilized during cidaroid mesenchyme specification. In contrast with hesC, the expression patterns and functions of alx1, tbr and ets1 were similar between P. baculosa and euechinoids. Based on these results, we propose that the roles of alx1, tbr and ets1 in mesenchyme specification were established in the common ancestor of echinoids, and that the DNG system was acquired in the euechinoid lineage after divergence from the cidaroid ancestor. The evolutionary timing of the establishment of the DNG suggests that the DNG was originally related to micromere and/or primary mesenchyme cell formation but not to skeletogenic cell differentiation.

"Micromere" formation and expression of endomesoderm regulatory genes during embryogenesis of the primitive echinoid Prionocidaris baculosa.

Blastomere composition and expression profiles of wnt8 and hox11/13b orthologues were examined in the primitive indirect-developing echinoid Prionocidaris baculosa. We found that blastomere composition in the 16-cell-stage Prionocidaris embryos was different from that of the indirect-developing echinoids belonging to Euechinoidea, a derived group of the echinoids. The sizes of the blastomeres in the 16-cell-stage embryo varied, and no embryos formed a "micromere quartet," a group of four equal-sized micromeres. The smallest blastomere was usually located around the vegetal pole. We also found significant differences in early expression profiles of wnt8 orthologues of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of wnt8 orthologue of Prionocidaris was not detected at the 16-cell stage; it began at the 32-cell stage in the broad area containing the vegetal pole. However, in later stages, the expression profiles of hox11/13b and wnt8 orthologues of Prionocidaris were similar to that of euechinoid orthologues. The present study suggests that there are considerable differences between Prionocidaris and euechinoids in early developmental mechanisms in the vicinity of the vegetal pole.

Role of the nanos homolog during sea urchin development.

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants.

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 modification of specification for the endomesoderm in the direct developing echinoid Peronella japonica: loss of the endomesoderm-inducing signal originating from micromeres.

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral-aboral (O-Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O-Ab axis.

Expression patterns of wnt8 orthologs in two sand dollar species with different developmental modes.

Two wnt8 orthologs, Smwnt8 and Pjwnt8, were isolated from an indirect developing sand dollar, Scaphechinus mirabilis, and a direct developing sand dollar, Peronella japonica, respectively. The expression patterns of two genes during early development were examined by whole mount in situ hybridization. The expression of Smwnt8 was initiated in the micromeres at the late 16-cell stage and expanded at the 64-cell stage to the whole vegetal hemisphere, including the presumptive endomesodermal regions. The timing of the initiation of Pjwnt8 transcription in the presumptive endomesoderm region was delayed by 2-3 cell cycles compared to that of Smwnt8. The delay, or molecular heterochrony, of Pjwnt8 transcription strongly suggests the existence of a substantial evolutionary change in the early endomesodermal specification of P. japonica. In addition to the endomesodermal expression during early embryogenesis, bilateral expressions were observed commonly in the ectoderm of two sand dollar species during larval stages.

cis-Regulatory inputs of the wnt8 gene in the sea urchin endomesoderm network.

Expression of the wnt8 gene is the key transcriptional motivator of an intercellular signaling loop which drives endomesoderm specification forward early in sea urchin embryogenesis. This gene was predicted by network perturbation analysis to be activated by inputs from the blimp1/krox gene, itself expressed zygotically in the endomesoderm during cleavage; and by a Tcf1/beta-catenin input. The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes beta-catenin nuclearization. Here, the modular cis-regulatory system of the wnt8 gene of Strongylocentrotus purpuratus was characterized functionally, and shown to respond to blockade of both Blimp1/Krox and Tcf1/beta-catenin inputs just as does the endogenous gene. The genomic target sites for these factors were demonstrated by mutation in one of the cis-regulatory modules. The Tcf1/beta-catenin and Blimp1/Krox inputs are both necessary for normal endomesodermal expression mediated by this cis-regulatory module; thus, the genomic regulatory code underlying the predicted signaling loop thus resides in the wnt8 cis-regulatory sequence. In a second regulatory region, which initiates expression in micromere and macromere descendant cells early in cleavage, Tcf1 sites act to repress ectopic transcription in prospective ectoderm cells.

Molecular heterotopy in the expression of Brachyury orthologs in order Clypeasteroida (irregular sea urchins) and order Echinoida (regular sea urchins).

The expression patterns of Brachyury (Bra) orthologs in the development of four species of sand dollars (order: Clypeasteroida), including a direct-developing species, and of a sea urchin species (order: Echinoida) were investigated during the period from blastula to the pluteus stage, with special attention paid to the relationship between the expression pattern and the mode of gastrulation. The sand dollar species shared two expression domains of the Bra orthologs with the Echinoida species, in the vegetal ring (the first domain) and the oral ectoderm (the second domain). The following heterotopic changes in the expression of the Bra genes were found among the sand dollar species and between the sand dollars and the Echinoida species. (1) The vegetal ring expressing Bra in the sand dollars was much wider and was located at a higher position along the AV axis, compared with that in the Echinoida species. The characteristic Bra expression in the vegetal ring of the sand dollar embryos was thought to be involved in the mode of gastrulation, in which involution continues from the beginning of invagination until the end of gastrulation. (2) Two of the three indirect-developing sand dollar species that were examined exhibited a third domain, in which Bra was expressed on the oral side of the archenteron. (3) In the direct-developing sand dollar embryos, Bra was expressed with an oral-aboral asymmetry in the vegetal ring and with a left-right asymmetry in the oral ectoderm. In the Echinoida species, Bra was expressed in the vestibule at the six-armed pluteus stage.

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

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

SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis.

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression.