Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.     

Molecular analysis of heterochronic changes in the evolution of direct developing sea urchins

The sea urchin Heliocidaris erythrogramma is a direct developer; it progresses directly from the gastrula to the juvenile adult without forming a pluteus larva. No larval skeleton is formed by mesenchyme cells, but formation of the juvenile skeleton is accelerated. We have examined two alterations in mesenchyme cell behavior that accompany this striking change in developmental pattern. 1) Rapid cell proliferation produces 1700–2200 mesenchyme cells by mid-gastrula, compared to 30–60 primary mesenchyme cells in species with typical larval development. This change may reflect the accelerated production of adult structures in H. erythrogramma. 2) B2C2 is a monoclonal antibody that recognizes primary (Anstrom et al., 1987) and adult mesenchyme cells associated with skeleton formation in typical developers. The altered pattern of B2C2 staining in H. erythrogramma (e.g., a later initial appearance of the B2C2 antigen) suggests that H. erythrogramma has deleted part of a larval program of development and accelerated its adult program of development. These results indicate that cellular and molecular heterochronies accompany the morphological changes in H. erythrogramma development.     

Par6 regulates skeletogenesis and gut differentiation in sea urchin larvae

Partitioning-defective (par) genes were originally identified as genes that are essential for the asymmetric division of the Caenorhabditis elegans zygote. Studies have since revealed that the gene products are part of an evolutionarily conserved PAR-atypical protein kinase C system involved in cell polarity in various biological contexts. In this study, we analyzed the function of par6 during sea urchin morphogenesis by morpholino-mediated knockdown and by manipulation swapping of the primary mesenchyme cells (PMCs). Loss of Par6 resulted in defects in skeletogenesis and gut differentiation in larvae. Phenotypic analyses of chimeras constructed by PMC swapping showed that Par6 in non-PMCs is required for differentiation of archenteron into functional gut. In contrast, Par6 in both PMCs and ectodermal cells cooperatively regulates skeletogenesis. We suggest that Par6 in PMCs plays an immediate role in the deposition of biomineral in the syncytial cable, whereas Par6 in ectoderm may stabilize skeletal rods via an unknown signal(s).     

Signals from primary mesenchyme cells regulate endoderm differentiation in the sea urchin embryo

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm-specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.     

Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos

Pigment cell precursors in the vegetal plate of late mesenchyme blastulae of the sea urchin Strongylocentrotus purpuratus begin to express a cell surface epitope recognized by the monoclonal antibody SP-1/20.3.1. When one-quarter gastrulae are dissociated into ectodermal and mesenchymal fractions, most SP-1/20.3.1 immunoreactive cells separate into the mesenchymal fraction, whereas at the full gastrula and all later stages almost all epitope-bearing cells are in the ectodermal fraction. Exposure of embryos to sulfate-free seawater p-nitrophenyl beta-D-xyloside, and tunicamycin, all of which prevent primary mesenchyme migration, does not inhibit SP-1/20.3.1 immunoreactive cells from distributing similarly to those in controls, although pigment synthesis is completely inhibited in sulfate-free conditions. Time-lapse video sequences reveal that pigment cells, and a small set of rapidly migrating, SP-1/20.3.1 immunoreactive amoeboid cells that appear in the pluteus, remain closely associated with the ectodermal epithelium during most of larval development. Transmission electron microscopy observations of plutei show pigment cells tightly apposed to the ectodermal epithelium at discontinuities in the basal lamina and sandwiched between the basal lamina and the epithelial cells. It is concluded that SP-1/20.3.1 immunoreactive mesenchymal cells invade the ectodermal epithelium and may use migratory substrates other than those used by primary mesenchymal cells.     

Development of the turtle carapace: Implications for the evolution of a novel bauplan

The chelonian carapace is composed of the endochondral ribs and vertebrae associated with a specialized dermis. The ribs are found in an aberrant position compared to those of all other tetrapods; they are superficial and dorsal to the limb girdles. This morphological arrangement, which constitutes the unique chelonian Bauplan, is examined from a developmental perspective. Embryos of Chelydra serpentina were studied during stages of carapace development. Tissue morphology, autoradiography, and indirect immunofluorescent localization of adhesion molecules indicate that the outgrowth of the embryonic carapace occurs as the result of an epithelial–mesenchymal interaction in the body wall. A carapacial ridge composed of mesenchyme of the dermis and overlying ectoderm is formed dorsal to the ectodermal boundary between somitic and lateral plate mesoderm. It is the anlage of the carapace margin, in which the ribs will eventually terminate. The ectoderm of the carapacial ridge is thickened into a pseudostratified columnar epithelium, which overlies a condensation in the mesenchyme of the dermis. Patterns of cell proliferation and the distribution of N-CAM and fibronectin in the carapacial ridge are consistent with patterns seen in other structures initiated by epithelial–mesenchymal interactions such as feathers and limb buds. Based on an analogy to this developmental mechanism in the development of the limb skeleton, a further analogy with the evolution of the limbs from lateral fin folds is used to form a hypothesis on the evolution of the carapace from elements of the primitive reptilian integument.     

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Cell differentiation during the larval development of the ophiuroid <Emphasis Type="Italic">Amphipholis kochii</Emphasis> Lütken, 1872 (Echinodermata: Ophiuroidea)

The differentiation of the ectodermal, entodermal, and mesodermal cell lines in developing plutei of the ophiuroid Amphipholis kochii was examined using electron microscopy and the immunochemical staining technique. The ectodermal cells form the pseudostratified epithelium of the ciliary band, the flattened epithelium of the body wall, and the esophageal epithelium. The epithelium of the ciliary band consists of ciliated and mucous cells; at its base is an axonal tract formed of the processes of neurons. The serotoninergic neurons form two lateral ganglia located along the paraoral ciliary band and the posterolateral arms’ ciliary band. The prominent features of the neurons are large size, the presence of a cilium, an electron-light cytoplasm filled with microvesicles with neurotransmitters, and a large nucleus with a predominant euchromatin. The ectoderm cells (except mucous cells) are characterized by the presence of a cilium surrounded by a collar of microvilli and a thin layer of apical extracellular matrix. The entodermal cells form the digestive tract epithelium and differentiate into four cell types: type I and II cells probably function in the nutrient uptake and assimilation; type III cells perhaps secrete digestive enzymes; and myoepithelial cells that constitute the cardiac and pyloric sphincters and the anus. Sclerenchymatous cells, which are the descendants of the primary mesenchyme, form a syncytium around the developing spicules. The biomineralization process is intrasyncytial, the ophioplutei spicules retain the cytoplasmic covering throughout the period of larval development. The secondary mesenchyme gives rise to smooth muscle cells and amebocytes. Muscle cells compose the circumesophageal musculature, the cell processes of each “muscle band” seem to fuse together. At the base of the preoral band are two symmetrically located groups of muscles, viz., the anterior dilators. Amebocytes function in excretion either near the epidermis or are able to penetrate through the epidermis and excrete wastes into the external environment. The mesoderm formed by the enterocoely gives rise to three pairs of coeloms; their cells remain unspecialized during the entire period of larval development. Results of this study are compared with the micro- and neuroanatomy of the larvae of other echinoderms.     

Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells.

Secondary mesenchyme in sea urchin embryos is released into the blastocoel after primary mesenchyme, and although these cells have been recognized for some time, we lack knowledge about many fundamental aspects of their origin and fate. Here we documented the ontogeny of one of the principal, and least well-known, types of cells derived from secondary mesenchyme. The blastocoelar cells arise from mesenchyme released from the tip of the archenteron following the initial phase of gastrulation. The cells migrate with their cell bodies suspended in the blastocoel, rather than being apposed to the basal lamina like primary mesenchyme. The cells extend numerous fine filopodia to form a network of cytoplasmic processes around the gut, along the skeletal rods, and within the larval arms. Once the network is formed, the cells maintain their positions, although they actively translocate vesicles and cytoplasm along their filopodia. Cell counts indicate there is an initial recruitment of cells during gastrulation, followed by a more gradual increase in cell number after the larva begins to feed. Lineage studies in which 16-cell-stage macromeres were injected with horseradish peroxidase indicate that almost all of the macromere-derived mesenchyme forms pigment cells and blastocoelar cells. We propose that blastocoelar cells are a distinct subset of secondary mesenchyme that forms fibroblast-like cells in the blastocoel of sea urchin embryos.     

Local cell interactions and the control of gastrulation in the sea urchin embryo

The sea urchin embryo is a good model system for studying the role of mechanical and cell-cell interactions during epithelial invagination, cell rearrangement and mesenchymal patterning in the gastrula. The mechanisms underlying the initial invagination of the archenteron have been surprisingly elusive; several possible mechanisms are discussed. In contrast to its initial invagination, the cellular basis for the elongation of the archenteron is better understood: both autonomous epithelial cell rearrangement and further rearrangement driven by secondary mesenchyme cells appear to be involved. Experiments indicate that patterning of freely migrating primary mesenchyme cells and secondary mesenchyme cells residing in the tip of the archenteron relies to a large extent on information resident in the ectoderm. Interactions between cells in the early embryo and later cell-cell interactions are both required for the establishment of ectodermal pattern information. Surprisingly, in the case of the oral ectoderm the fixation of pattern information does not occur until immediately prior to gastrulation.     

In vitro fusion and separation of sea urchin primary mesenchyme cells

Time-lapse videomicroscopy of cultured primary mesenchyme cells from mesenchyme blastulae of the sea urchin Lytechinus pictus demonstrates the dramatic ability of these cells to undergo cell fusion and cell separation. Although this plasticity of cell associations is presumed to play a role in the formation of the syncytial cables that secrete the larval skeleton, the surfaces of these cells must be specialized for fusion and cell separation.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.     

Occurrence of two NOS isoforms in the developing gut of sea bass Dicentrarchus labrax (L.)

In this work we have examined the appearance and distribution of nitric oxide synthase (NOS), with histochemical, immunohistochemical and biochemical methods, during development of the sea bass (Dicentrarchus labrax) gut. The data showed that both the calcium-calmodulin dependent neuronal isoform (nNOS) and calcium-independent inducible isoform (iNOS) are present in the larval gut of sea bass. The nNOS-immunoreactivity was present in the epithelial cells and enteric nerve cells of gut both in the 8-day-old specimens and in the 24-day-old-larvae. In the adult nNOS-immunoreactivity disappeared from epithelial cells, remaining in the wall intramural neurons and fibers. The iNOS-immunoreactivity was present in the epithelial cells of 24-day-old-larvae and was not detectable in the adult gut. Western blot analysis and determination of NOS activity also demonstrated the presence of the two NOS isoforms, nNOS and iNOS, in the gut of 24-day-old specimens. The presumably different roles played by the two isoforms of enzyme are discussed. The presence of nNOS isoform in the gut enteric neurons of the same larval stages of D. labrax in which we previously demonstrated the presence of substance P and Vasoactive Intestinal Polypeptide (VIP), may suggest that all these three components of the motility control system are already present in the larval phase. Nitric oxide (NO) may be also involved in the early immune response. The present results on the occurrence of iNOS isoform in epithelial gut cells of the same regions in which the gut-associated lymphoid tissue (GALT) will differentiate, may suggest for NO a role in early defence mechanisms, before the establishment of immune responses in GALT. Finally, the developmental and regional differences in nNOS and iNOS expression also suggest a regulatory role in development and differentiation of the sea bass gut.     

Ectodermal Wnt signaling regulates abdominal myogenesis during ventral body wall development

Defects of the ventral body wall are prevalent birth anomalies marked by deficiencies in body wall closure, hypoplasia of the abdominal musculature and multiple malformations across a gamut of organs. However, the mechanisms underlying ventral body wall defects remain elusive. Here, we investigated the role of Wnt signaling in ventral body wall development by inactivating Wls or β-catenin in murine abdominal ectoderm. The loss of Wls in the ventral epithelium, which blocks the secretion of Wnt proteins, resulted in dysgenesis of ventral musculature and genito-urinary tract during embryonic development. Molecular analyses revealed that the dermis and myogenic differentiation in the underlying mesenchymal progenitor cells was perturbed by the loss of ectodermal Wls. The activity of the Wnt-Pitx2 axis was impaired in the ventral mesenchyme of the mutant body wall, which partially accounted for the defects in ventral musculature formation. In contrast, epithelial depletion of β-catenin or Wnt5a did not resemble the body wall defects in the ectodermal Wls mutant. These findings indicate that ectodermal Wnt signaling instructs the underlying mesodermal specification and abdominal musculature formation during ventral body wall development, adding evidence to the theory that ectoderm-mesenchyme signaling is a potential unifying mechanism for the origin of ventral body wall defects.