P16 is an essential regulator of skeletogenesis in the sea urchin embryo

The primary mesenchyme cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that culminates in the formation of the larval endoskeleton. Recent studies have identified components of a gene regulatory network that underlies PMC specification and differentiation. In previous work, we identified novel gene products expressed specifically by PMCs (Illies, M.R., Peeler, M.T., Dechtiaruk, A.M., Ettensohn, C.A., 2002. Identification and developmental expression of new biomineralization proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Genes Evol. 212, 419-431). Here, we show that one of these gene products, P16, plays an essential role in skeletogenesis. P16 is not required for PMC specification, ingression, migration, or fusion, but is essential for skeletal rod elongation. We have compared the predicted sequences of P16 from two species and show that this small, acidic protein is highly conserved in both structure and function. The predicted amino acid sequence of P16 and the subcellular localization of a GFP-tagged form of the protein suggest that P16 is enriched in the plasma membrane. It may function to receive signals required for skeletogenesis or may play a more direct role in the deposition of biomineral. Finally, we place P16 downstream of Alx1 in the PMC gene network, thereby linking the network to a specific “effector” protein involved in biomineralization.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo

During sea urchin embryogenesis, the skeleton is produced by primary mesenchyme cells (PMCs). PMCs undergo a sequence of morphogenetic behaviors that includes ingression, directed migration, and cell–cell fusion. Ultimately, PMCs deposit the calcite-containing biomineral that forms the endoskeleton of the late embryo and early larva. The endoskeleton has a stereotypical structure and is the major determinant of the distinctive, angular shape of the larva. Although many candidate biomineralization proteins have been identified, functional data concerning these proteins are scant. Here, we identify and characterize two new biomineralization genes, p58-a and p58-b. We show that these two genes are highly conserved in Strongylocentrotus purpuratus and Lytechinus variegatus, two sea urchin species whose ancestors diverged approximately 100 mya. The p58-a and p58-b genes lie in tandem on the chromosome, suggesting that one of the two genes arose via a gene duplication event. The two genes encode closely related, type I transmembrane proteins. We have established by whole mount in situ hybridization that p58-a and p58-b are expressed specifically in the PMCs in both species. Knockdown of either gene by morpholino antisense oligonucleotides leads to profound defects in skeletogenesis, although skeletal elements are not completely eliminated. The P58-A and P58-B proteins do not appear to play a role in the specification, directed migration or differentiation of the PMCs, but most likely are directly involved in biomineralization during sea urchin embryonic development.     

Isolation of sea urchin embryo cell surface membranes on polycationic beads

Summary Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [³H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na⁺/K⁺ ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.     

Specification and differentiation processes of secondary mesenchyme-derived cells in embryos of the sea urchin Hemicentrotus pulcherrimus

Four types of mesoderm cells (pigment cells, blastocoelar cells, coelomic pouch cells and circumesophageal muscle cells) are derived from secondary mesenchyme cells (SMC) in sea urchin embryos. To gain information on the specification and differentiation processes of SMC-derived cells, we studied the exact number and division cycles of each type of cell in Hemicentrotus pulcherrimus. Numbers of blastocoelar cells, coelomic pouch cells and circumesophageal muscle fibers were 18.0 +/- 2.0 (36 h post-fertilization (h.p.f.)), 23.0 +/- 2.5 (36 h.p.f.) and 9.5 +/- 1.3 (60 h.p.f.), respectively, whereas the number of pigment cells ranged from 40 to 60. From the diameters of blastocoelar cells and coelomic pouch cells, the numbers of division cycles were elucidated; these two types of cells had undertaken 11 rounds of cell division by the prism stage, somewhat earlier than pigment cells. To determine the relationship among the four types of cells, we tried to alter the number of pigment cells with chemical treatment and found that CH3COONa increased pigment cells without affecting embryo morphology. Interestingly, the number of blastocoelar cells became smaller in CH3COONa-treated embryos. In contrast, blastocoelar cells were markedly increased with NiCl2 treatment, whereas the number of pigment cells was markedly decreased. The number of coelomic pouch cells and circumesophageal muscle fibers was not affected with these treatments, indicating that coelomic pouch and muscle cells are specified independently of, or at much later stages, than pigment and blastocoelar cells.     

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.     

The dynamics of secretion during sea urchin embryonic skeleton formation

Skeleton formation involves secretion of massive amounts of mineral precursor, usually a calcium salt, and matrix proteins, many of which are deposited on, or even occluded within, the mineral. The cell biological underpinnings of this secretion and subsequent assembly of the biomineralized skeletal element is not well understood. We ask here what is the relationship of the trafficking and secretion of the mineral and matrix within the primary mesenchyme cells of the sea urchin embryo, cells that deposit the endoskeletal spicule. Fluorescent labeling of intracellular calcium deposits show mineral precursors are present in granules visible by light microscopy, from whence they are deposited in the endoskeletal spicule, especially at its tip. In contrast, two different matrix proteins tagged with GFP are present in smaller post-Golgi vesicles only seen by electron microscopy, and the secreted protein are only incorporated into the spicule in the vicinity of the cell of origin. The matrix protein, SpSM30B, is post-translationally modified during secretion, and this processing continues after its incorporation into the spicule. Our findings also indicate that the mineral precursor and two well characterized matrix proteins are trafficked by different cellular routes.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Fibronectin-binding acid polysaccharide in the sea urchin embryo

Summary A novel fibronectin-binding acid polysaccharide (FAPS) was isolated from embryos of the sea urchin. Binding of FAPS to fibronectin was quantitatively measured at physiological pH and ionic strength by two different assay systems. Immunofluorescent studies revealed that FAPS is localized in the extracellular matrix surrounding the mesenchyme cells and primitive gut of middle gastrula. Sea urchin fibronectin was also detected in the extracellular matrix surrounding mesenchyme cells and the cells surrounding the blastopore. When a monoclonal antibody to FAPS (anti-FAPS) was microinjected into the blastocoel, more than one pair of triradiate spicular rudiments was formed and the malformation of spicules was induced. Armless and deformed larvae were also induced by anti-FAPS. FAPS may regulate the number, length, position and direction of spicules. These results implicate the extracellular matrix of the blastocoel in the complex process of differentiation of mesenchyme and the formation of spicules.     

Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae

Summary The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching

Summary The early sea urchin embryo is supported in a concentric position within the perivitelline space by elongated microvilli which are attached to the fertilization envelope by extracellular matrix fibers. This “attachment complex,” of microvillus tip: extracellular matrix fibers: fertilization envelope, was revealed by two methods: the use of pronase or calcium-free sea water to dissolve the extracellular matrix fibers, thus causing the eggs to lose their concentric location, and the visualization of the “attachment complex” using video-enhanced differential interference contrast microscopy and transmission electron microscope images. The presence of the “attachment complex” helps in understanding two types of early developmental events: (1) the apparently continual change in microvillus length during cleavage stages which retains the embryos in their concentric position and (2) the hatching process.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Comparative in vivo evaluation of polyalkoxy substituted 4H-chromenes and oxa-podophyllotoxins as microtubule destabilizing agents in the phenotypic sea urchin embryo assay

A series of polyalkoxy substituted 7-hydroxy- and 7-methoxy-4-aryl-4H-chromenes were evaluated using the sea urchin embryo model to yield several compounds exhibiting potent antimitotic microtubule destabilizing activity. Data obtained by the assay were further confirmed in the NCI60 human cancer cell screen. The replacement of methylenedioxy ring A and lactone ring D in podophyllotoxin analogues by 7-methoxy, 2-NH₂, and 3-CN groups in 4-aryl-4H-chromenes resulted in potent antimitotic microtubule destabilizing agents. Feasible synthesis and high yields render 7-methoxy-4H-chromenes to be a promising series for further anticancer drug development.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.