Initial stages of calcium uptake and deposition in sea urchin embryos

Netta Vidavskya, Sefi Addadib, Julia Mahamidc, Eyal Shimonid, David Ben-Ezrae, Muki Shpigele, Steve Weinera, and Lia Addadia,1

aDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; bB-nano Ltd., Rehovot 76326, Israel; cDepartment of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; dDepartment of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel; and eIsrael Oceanographic and Limnological Research, National Center for Mariculture, Eilat 88112, Israel

Edited by Patricia M. Dove, Virginia Polytechnic Institute and State University, Blacksburg, VA, and approved October 31, 2013 (received for review July 10, 2013) Sea urchin larvae have an endoskeleton consisting of two calcitic fluorescent signal at the spicule tip (13). The dynamics of spicule spicules. We reconstructed various stages of the formation elongation also was studied in vivo by using calcein, which la- pathway of calcium carbonate from calcium ions in sea water to beled the surface of newly formed spicule areas (7, 14). Blocking mineral deposition and integration into the forming spicules. or interfering with calcium uptake by the cells, both in vivo and Monitoring calcium uptake with the fluorescent dye calcein shows in PMC cultures, shows that for the spicule to be formed, cal- that calcium ions first penetrate the embryo and later are de- cium first has to penetrate the cells (15–17). The manner in posited intracellularly. Surprisingly, calcium carbonate deposits are which calcium is transported to the PMCs is not known (7). It is distributed widely all over the embryo, including in the primary known that the ectoderm cells take part in spicule mineraliza- mesenchyme cells and in the surface epithelial cells. Using cryo- tion, especially in determining the placement of PMCs, which in SEM, we show that the intracellular calcium carbonate deposits turn specify the morphology of the spicule. The involvement of are contained in vesicles of diameter 0.5–1.5 μm. Using the newly ectoderm cells in calcium transport to the PMCs is not well developed airSEM, which allows direct correlation between fluo- understood, considering the fact that PMC cultures produce rescence and energy dispersive spectroscopy, we confirmed the spicules in the absence of ectoderm cells if growth factors are presence of solid calcium carbonate in the vesicles. This mineral introduced (8, 14, 18). phase appears as aggregates of 20–30-nm nanospheres, consistent Beniash et al. (19) showed that the PMCs contain electron- with amorphous calcium carbonate. The aggregates finally are in- dense granules of amorphous calcium carbonate (ACC) about troduced into the spicule compartment, where they integrate into 0.5–1.5 μm in diameter. These granules were assumed to in- the growing spicule. tegrate into the growing spicule after fusion with the syncytium membrane. The initial crystalline deposit in the syncytium is in | mineralization pathway | sea urchin embryonic spicule | the form of a rhombohedral crystal (20). The spicule transient precursor mineral phase | intracellular mineral deposition grows by the addition of transient ACC, which then partially CHEMISTRY transforms into calcite through secondary nucleation (21, 22). uring the biomineralization process, ions from the envi- The transformation from ACC to crystalline calcite is complex. Dronment are transported to the mineralization site by using Examination of the spicule surfaces with X-ray absorption different strategies, such as cell penetration through specific ion spectroscopy using extended X-ray absorption fine structure channels (1), delivery by vesicles (2), and formation of a pre- (EXAFS) and X-ray photoelectron emission microscopy (X- cursor amorphous mineral phase (3). The intracellular formation PEEM) showed three distinct mineral phases present in adjacent of a transient amorphous phase enables the mineral to be delivered sites at a scale of tens of nanometers. The initial phase, ACC1, is short-lived and presumably a hydrated ACC phase. The second

efficiently to the mineralization site in a concentrated manner, thus BIOPHYSICS AND overcoming limitations of ion transport (4). phase, ACC2, is an intermediate transient form of ACC, whereas COMPUTATIONAL BIOLOGY The formation of calcite spicules by the embryonic sea urchin the third phase is crystalline calcite (21, 23). is a fascinating and well-documented example of a complex mul- tistage biomineralization process. Sea urchin embryos obtain cal- Significance cium directly from sea water until they can feed (5). The calcium is transported from the sea water through various outer tissue layers With the onset of gastrulation, sea urchin embryos deposit and is deposited inside cells. This mineral is then translocated to a calcium carbonate endoskeleton consisting of two spicules. a delimited space inside a syncytium in which an endoskeleton Sea water is the source for the mineral ions, but the specific composed of two spicules, each of which diffracts X-rays as stages of the transport and deposition pathway are not well a single crystal of calcite, is formed (6). The cells responsible for understood. This study shows that the first-formed mineral is the formation of the syncytium are the primary mesenchyme cells deposited inside intracellular micrometer-size vesicles as solid (PMCs). The spicules mineralize within the syncytium space, nanospheres. Surprisingly, the initial deposits are distributed enveloped by a thin extracellular layer (7). The PMCs are be- widely inside the embryonic cells, including epithelial cells. The lieved to control the entire biomineralization process, because possibility that the whole embryo is geared toward depositing spicule formation occurs in isolated and cultured PMCs (8). In mineral for spicule formation or other purposes, may contribute the embryo, however, the calcium and carbonate ions from the to the understanding of biomineralization processes in general. sea water have to pass through various outer tissue layers. The Author contributions: N.V., S.W., and L.A. designed research; N.V., S.A., J.M., and E.S. cells of these tissues thus control the ultrastructural and chemical performed research; S.A., E.S., D.B.-E., and M.S. contributed new reagents/analytic tools; milieu in which mineralization by the PMCs occurs and are also N.V., S.A., S.W., and L.A. analyzed data; and N.V., S.W., and L.A. wrote the paper. involved in the process. The authors declare no conflict of interest. Calcein is a small fluorescent molecule that may be used to This article is a PNAS Direct Submission. label calcium ions (9–12). Calcium ion labeling experiments with 1To whom correspondence should be addressed. E-mail: [email protected]. calcein in PMC cultures first demonstrated the formation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. calcium granules inside PMCs, followed by the appearance of a 1073/pnas.1312833110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1312833110 PNAS | January 7, 2014 | vol. 111 | no. 1 | 39–44 Downloaded by guest on September 27, 2021 Although it is known that the spicules form through secondary nucleation of ACC, many stages of the mineralization pathway are not clear. Some of the open questions are fundamental for understanding spicule mineralization, namely, what is the cal- cium pathway from sea water to the syncytium, do ACC deposits also form in cells that are not PMCs, and how are the ACC deposits transported into the spicule compartment? To address these questions, we studied the calcium pathways in whole embryos. Results SEM imaging under cryogenic conditions was performed on sea urchin embryos and larvae at different stages post fertilization. Fig. 2. Cryo-SEM micrograph of a high-pressure frozen and freeze-frac- The sample preparation procedure involves only high-pressure tured sea urchin embryo at the gastrula stage showing the spicule (S) and adjacent mineral-bearing vesicles, packed with nanospheres (arrowheads). freezing and freeze fracture of unfixed and unstained embryos. BSE image in Fig. S3. Cryo-SEM allows high-resolution imaging of biological samples as close as possible to their native state, minimizing artifacts caused by chemical procedures and drying. A cryo-SEM image of were observed after 1 h (Fig. 3 A and D). The calcein label is A a sea urchin embryo at the late gastrula stage (Fig. 1 ) shows the observed as a disperse cloud confined mainly within the blasto- layer of epithelial cells, the invaginated archenteron, and the rel- coel. After a longer calcein pulse (40 min), the calcein label atively large cell free space called the blastocoel. In the blastocoel, appears as concentrated granules inside the epithelial cells and specialized PMCs are interconnected in a syncytium where the possibly the PMCs and other cells (Fig. 3 B and E). spicules are formed. Two fractured spicules (backscattered image A Surprisingly, when embryos were developed from fertilization in Fig. S1 ) surrounded by PMCs are visible within the blastocoel continuously inside calcein-labeled sea water, calcein was de- on the two sides of the invaginated archenteron. Of particular tected as concentrated micrometer-sized granules all over the interest is the presence of a multivesicular body adjacent to the embryo (Fig. 3 C and F). The fluorescent label is concentrated PMCs (Fig. 1A and Fig. S1C). not only in the blastocoel, where it presumably is associated with Cryo-SEM observations of PMCs show intracellular deposits the PMCs attached to the forming spicules, but also in the epi- enclosed by membrane-delimited vesicles ranging in size from 0.5 to 1.5 μm. These vesicles contain densely packed nanospheres thelial cells. When chemically fixed embryos were exposed to of 20–30 nm, reminiscent of the nanospheres of ACC observed in calcein solution under the same conditions as those of the live the forming spicule (24) (Fig. 1 B and C). Similar vesicles also embryos, calcein was observed inside the embryo, mostly as a are observed crossing the boundary between the cell and the dispersed cloud (Fig. S4). No label was observed in the spicule, spicule compartment, and in the proximity of the spicule (Fig. 2 and no extensive granular labeling occurred inside the cells. It and Fig. S2). The nanospheres produce a positive backscattered should be taken into account that the labeling or absence thereof electron (BSE) signal, which implies that they are composed of in the fixed embryo may be influenced by the fixation procedure a condensed phase with high electron density (Figs. S1B and S3). and thus may not exclusively reflect passive label diffusion in vivo. Because of the three-dimensionality of the sample surface, In agreement with the calcein labeling, cryo-SEM from un- however, a positive BSE signal may be deceptive, and alone does treated embryos reveals the presence of abundant nanosphere- not provide proof of the electron-dense nature of the particles. containing vesicles also in the epithelial cells (Fig. 4A). The We therefore carried out calcein pulse–chase experiments to nanospheres contained in the vesicles inside the epithelial cells obtain a 3D overview of the pathway followed by the calcium (Fig. 4 B–D) are within the same size range as those observed in ions and possibly to identify mineral-bearing vesicles within the PMCs. the PMCs. A definitive demonstration still is required to show that the Localization of calcium in the embryonic environment was calcein-labeled granules indeed are composed of solid calcium carried out using a confocal microscope monitoring calcein fluo- carbonate deposits. In particular, we need to demonstrate that rescence. Embryos at the late gastrula stage first were exposed to calcein specifically maps the distribution of calcium and not of a short 10-min pulse of calcein dissolved in the sea water and other divalent metal ions that also bind to calcein, such as Zn

Fig. 1. Cryo-SEM micrograph of a high-pressure frozen and freeze-fractured sea urchin embryo at the late gastrula stage. (A) The spicule cross-sections are marked with arrows. The archenteron (Ar) and epithelial cells (Ep) of the embryo are fractured, showing large numbers of intracellular vesicles of 0.5–1.5 μm. Note the multivesicular body adjacent to the PMC (arrowhead), enlarged in Fig. S1C. The blastocoel is marked with an asterisk. (B) The spicule cross-section (S; BSE image of the spicule area in Fig. S1B) is adjacent to a PMC that contains many vesicles, some empty and some packed with nanospheres (arrow). (C) Enlargement of the marked vesicle in B, showing nanospheres of 20–30 nm.

40 | www.pnas.org/cgi/doi/10.1073/pnas.1312833110 Vidavsky et al. Downloaded by guest on September 27, 2021 Fig. 3. Confocal micrographs of live calcein-labeled sea urchin embryos at the gastrula stage. (A–C) Green calcein fluorescence emission. (D and E) The fluorescence image in A and B is merged with the bright-field image. (F) Bright-field image of C.(A and D) The embryo received a 10-min calcein pulse at 40 h post fertilization (hpf), followed by a 1-h chase period in sea water. Calcein appears as a cloud in the blastocoel of the embryo and is not detected in an intracellular environment. One focal plane. (B and E) The embryo received a calcein pulse of 40 min at 40 hpf, followed by a 1-h chase period in sea water. Four focal planes, 2 μm apart, were stacked together. The calcein label is observed in the cellular environment in a more concentrated manner, in both the epithelial and other cells. (C and F) Embryo that was developed continuously in calcein-labeled sea water (46 hpf). Micrometer-size calcein-labeled granules are observed all over the embryo. One focal plane. Scale bars: 20 μm.

and Fe. With this in mind, we used the newly developed “airSEM” obtained from the section (Fig. 5C) with the distribution of heavy (B-nano Ltd.; www.b-nano.com), which is an SEM that operates in atoms using backscattered electrons (Fig. 5A), and with the the atmosphere at ambient conditions (the sample is not kept calcium map using energy dispersive spectroscopy (EDS) (Fig. under vacuum). Using this instrument, samples are shuttled di- 5B), all in the exact same area. A comparison of the images rectly from under the optical microscope objective to under the shows that the calcein signal is associated with calcium rather SEM column, which is separated from the atmospheric envi- than with other ions or with free calcein (Fig. 5D). The differ- ronment by a thin membrane that does not touch the sample. ences between the images mainly are a result of the fact that the CHEMISTRY Movement between the two optical axes is done in synchronous depth of sampling of the three techniques is different. Fluores- register so that the images obtained from the SEM can be cor- cence detects signals from the whole slice (100 μm), whereas related directly with images from a fluorescence microscope. EDS and backscattering under the conditions used detect signals Unlike traditional SEMs, the airSEM allows imaging of humid to a depth of up to about 3 μm. samples, with no need for dehydration. For further information To show that the calcium deposits are of solid calcium car- on the setup, see Materials and Methods. bonate, we again used the airSEM to image a 50-μm slice of a Continuously calcein-labeled, fixed, and sectioned embryos fixed embryo at higher resolution. A good correlation between embedded in a wet agarose gel produced in sea water first were the backscattered electron signal and the calcium EDS map was BIOPHYSICS AND

observed under the fluorescence microscope and then were observed (Fig. 6 A and C). Quantitative elemental EDS analyses COMPUTATIONAL BIOLOGY shuttled under the SEM objective at the precise location imaged were performed in two different areas within the same image in the light microscope. Fig. 5 compares the fluorescence image (Fig. 6C): a part of the spicule, which is composed of solid CaCO3,

Fig. 4. Cryo-SEM micrograph of a high-pressure frozen and freeze-fractured sea urchin embryo at the late gastrula stage. (A) The PMCs and epithelial cells (Ep) of the embryo are fractured, showing large numbers of intracellular vesicles (arrows). N, nuclei; S, spicule. (B) Epithelial cells, enlarged from A, showing fractured intracellular vesicles (arrow). (C and D) Fractured vesicle from an epithelial cell containing nanospheres (C) with corresponding BSE contrast image (D).

Vidavsky et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 41 Downloaded by guest on September 27, 2021 Table 1. EDS elemental analysis of the spicule and cell areas Element Spicule, wt % Cells, wt %

Oxygen 27 ± 12 35 ± 15 Carbon 18 ± 916± 8 Sodium 22.1 ± 5.4 18.4 ± 4.7 Chlorine 18.8 ± 2.4 12.4 ± 1.7 Nitrogen 6 ± 410± 6 Calcium 6.3 ± 0.7 6.2 ± 0.7 Magnesium 1.0 ± 0.3 0.9 ± 0.3 Phosphorus 0.08 ± 0.09 0.15 ± 0.10

We show that calcein-labeled calcium first appears dispersed inside the blastocoel, where the calcium most likely still is in the form of free ions. Calcium is later observed as concentrated micrometer-size intracellular granules, not only inside PMCs, which are known to play a crucial role in spicule mineralization, but also in epithelial cells. These granules are composed of solid calcium carbonate nanospheres that most likely are ACC. The calcitic spicule is composed of nanospheres of the same size and shape (24), indicating that the intracellular nanosphere-bearing Fig. 5. Correlative airSEM image of a ≤100-μm slice of a fixed embryo at granules are part of the mineralization pathway and eventually ambient conditions showing the BSE signal (A), calcium EDS map (B), and are integrated into the spicule. calcein fluorescence (C). (D) The correlation between the calcium EDS signal The fact that calcium is first observed dispersed in the blas- (B) and calcein fluorescence (C) is shown in the superimposition of the two images. Yellow, colocalized signals; green and red, fluorescence and calcium tocoel implies that calcium penetrates from sea water into the EDS signals, appearing separately. Slight deformation of the surface, re- embryo through or between the epithelial cells and subsequently sulting from electron beam damage, may have decreased the actual colo- is deposited intracellularly in the form of solid calcium carbonate calization area. deposits inside vesicles. Based on Beniash et al. (19), we assume that these intracellular calcium carbonate deposits are composed of ACC. Surprisingly, these mineral deposits are formed not only and a group of cells that because of their location near the outer in the PMCs, but also in epithelial cells. This raises the question: embryonic surface and the spicule, probably consist of PMCs and what is the function of the calcium carbonate deposits inside the epithelial cells. The amount of calcium measured in the spicule, epithelial cells? 6.3 wt %, is practically identical to the amount measured in the One possible function of the epithelial mineral deposits is that intracellular compartment, 6.2 wt % (Table 1; related EDS they are a reservoir of calcium and carbonate ions needed by the spectra are shown in Fig. S5). Control areas with a low calcein embryo for other purposes, or conceivably for future shell and label and high backscattering signal give calcium amounts <2% tooth formation after the larva undergoes metamorphosis. An- (Fig. S6). This observation shows that solid calcium carbonate is other possibility is that some of this mineral is a reservoir for present within the cells. At both locations, the calcium content is spicule formation. In this case, it would have to be transported lower than the expected calcium concentration of pure CaCO3 through the blastocoel to the syncytium, which might occur in the (40 wt %) because of the presence of other elements in the sample form of ions after the extruded ACC dissolves. An alternative area, especially Na and Cl from sea water and C and O from the possibility is that the granules might be transported as such agarose and the cell components. The calcium concentration through the blastocoel. We did observe multivesicular bodies measured in the cellular area is much higher than any value that (Fig. 1A and Fig. S1C) composed of vesicles of the same size as could be obtained from concentrated cytoplasm or sea water. the mineral-bearing intracellular vesicles. These unusual non- cellular structures may be transporting mineral from the epi- Discussion thelial cells to the syncytium, where spicule formation occurs. It We monitored aspects of the calcium pathway in sea urchin is interesting to note that Ettensohn and Malinda (25) demon- embryos from the sea water until incorporation in the spicules. strated in vivo that photoablation of a distinctive region of

Fig. 6. BSE image (white) and calcium EDS map (red) of a fixed and sliced embryo taken with the airSEM. (A) Whole embryo, with calcium EDS map (red) and BSE signal (white) superimposed. The spicule is marked with an arrow, and the membrane enveloping the embryo is marked with arrowheads. (B) En- largement of the square-labeled area in A, containing a part of the spicule (arrow) and cell group (arrowhead); BSE image. (C) The same region as in B, with calcium EDS map (red) superimposed. EDS quantitative analysis of the marked areas is presented in Table 1. C, cell group; S, spicule.

42 | www.pnas.org/cgi/doi/10.1073/pnas.1312833110 Vidavsky et al. Downloaded by guest on September 27, 2021 ectoderm resulted in inhibition of spicule elongation, suggesting polarized light. The research involving sea urchins is approved by the that a local ectoderm–PMC interaction is required for spicule Israel Oceanographic and Limnological Research, National Center for growth. Our results indicate that the interactions between the Mariculture. ectoderm cells and the spicule compartment also may involve μ – actual calcium transport. Calcein Labeling. Calcein, 150 g/mL (Sigma Aldrich), was dissolved in sea The PMCs presumably take up dissolved ions from the blas- water containing 30 mg/L penicillin and 15 mg/L streptomycin and filtered in a 0.22-μm sterile Corning filter system. For pulse–chase experiments, the tocoel. It has been shown in culture that they do possess calcium embryos were transferred from sea water to calcein-labeled sea water for 10 ion pumps (15). Adding manganese in vivo reduced calcium or 40 min (pulse period) at 40 h post fertilization, washed in sea water, and uptake into the PMCs and prevented spicule formation, possibly transferred to unlabeled sea water for 1 h (chase period). For continuous because of manganese and calcium competition on the same ion labeling, the embryos were developed from fertilization inside the calcein- pumps (16). Interestingly, calcium was detected as calcein-labeled labeled sea water at 18 °C with gentle shaking (100 rpm). Approximately intracellular granules even in Mn-exposed embryos (16). This 46 h post fertilization, the embryos were washed with sea water. might be the result of cellular calcium uptake that is not in the For the control experiment, embryos 46 h after fertilization were fixed form of ions, possibly through particle endocytosis. for 1 h in sea water containing 2.5% (wt/vol) glutaraldehyde and 2.5% We do not know how the mineral in the vesicles of the PMCs (wt/vol) paraformaldehyde. The fixed embryos were washed in sea water is translocated into the membrane-bound spicule formation com- and exposed to the same calcein solution as the live embryos for 40 min. partment. Using cryo-SEM, we have observed vesicles located in The embryos were washed with sea water and imaged in the confocal the interface between a PMC and the spicule compartment that microscope after a 1-h chase period in sea water. appear to be heading toward the spicule (Fig. S2). We also have A drop of the embryo suspension was put on a glass-bottom Petri plate, observed spicules in the process of being formed surrounded by followed by a drop of 5% (wt/vol) agarose gel (SeaKem) in sea water solution to confine the embryos spatially in a hydrated environment. micrometer-sized mineral granules resembling the intracellular granules (Fig. 2). These observations are consistent with the Confocal Imaging. The calcein fluorescence emission and transmitted light mineral granules being translocated in the solid state from inside bright-field images were obtained by using a confocal laser scanning mi- the cells into the spicule formation compartment, as suggested by croscope, Olympus FluoView 1000, with 20× and 40× objective lens magni- Beniash et al. (19) and Ingersoll et al. (26). fication. The laser wavelength was (excitation) 488 nm, and emission was Stable and transient amorphous mineral phases formed by taken with a 520–550-nm band-pass filter. A Z series of both the transmitted many organisms consist of nanospheres (27, 28). It is possible and fluorescence images was collected for each specimen with 2-μm steps. that the nanospheres themselves are derived from smaller building blocks, such as stable ion clusters, which were shown to exist even Cryo-SEM Imaging. Embryos during the late gastrula stage, ∼40 h post fer- in undersaturated calcium carbonate solutions (29, 30). In the case tilization, were high-pressure frozen: 10 μL of the embryo suspension was of the disordered calcium phosphate precursor phase in zebrafish sandwiched between two metal discs (3-mm diameter, 0.1-mm cavities) and fin rays, the nanospheres also appear in intracellular membrane- cryoimmobilized in a high-pressure freezing device (HPM10; Bal-Tec). The delimited vesicles, which are delivered to the mineralization site frozen samples were kept in liquid nitrogen and transferred by using (2). In vitro studies of the involvement of nanospheres in calcite a vacuum cryotransfer device (VCT 100; Leica Microsystems) to a freeze- fracture device (BAF 60; Leica Microsystems). Samples were freeze-fractured

single-crystal growth from biogenic ACC exposed to water show − − CHEMISTRY – at 120 °C, etched for 10 min at 105 °C, and coated with 2.5 nm platinum/ that 20 30-nm nanospheres are translocated to the surface of a carbon by double-axis rotary shadowing. Samples were observed at −120 °C growing crystal, where they crystallize (28). Analogously, in the in an Ultra 55 SEM (Zeiss) by using a secondary electron in-lens detector and case of the growing embryonic spicule, after delivery of the vesicle- a backscattered electron in-lens detector (5 kV). Image brightness and contrast packed mineral, the disaggregated nanospheres presumably then levels were adjusted by using Adobe Photoshop. crystallize as a result of contact with other already-crystalline nanospheres to ultimately form a single crystal (21, 22). airSEM Imaging. Embryos continuously developed in calcein-labeled sea water From the discussion above, a conceivable scenario for a general were chemically fixed using 4% paraformaldehyde in sea water for 1 h, ion pathway emerges. The ions first are concentrated, and the washed with sea water, and mounted in 7% agarose gel dissolved in sea mineral is deposited in both specialized and nonspecialized cells. water. The mounted sample was sliced using a vibratome to a thickness of 50– BIOPHYSICS AND The mineral then is transported to the mineralization site, where it 100 μm, and the slices were put on a glass slide and imaged using the airSEM COMPUTATIONAL BIOLOGY is incorporated as such into the developing mineralized tissue. within 2 h. The airSEM (B-nano Ltd.) operates in a direct correlative manner: the optical and SEM microscopes are located on a single platform, both in Conclusions upright geometry, and the sample is shuttled between the two optical axes with accurate registration. Embryos first were imaged under the optical We demonstrate using confocal microscopy, cryo-SEM, and microscope for sample orientation and fluorescent imaging. Epifluorescent airSEM that during the process of calcium carbonate deposition images of the calcein fluorescence (Ex’: BP460-495; Em’: BA510-550) were in sea urchin embryos, calcium is first introduced into the blas- acquired using a 20× objective. The sample then was shuttled to the optical tocoel in a dispersed form. The mineral then appears inside axes of the SEM, and the same area was imaged at a matching field of view PMCs, as well as inside epithelial cells, as membrane-bound by using backscattered electrons (beam energy: 30 kV; probe current: 1000 micrometer-size granules. The granules finally are introduced pA). It is noteworthy that working in air, the backscattered electron image into the spicule compartment, where they presumably disaggre- does not suffer from charge-induced problems, as occurs in conventional gate into nanospheres and crystallize by secondary nucleation. SEM microscopes operating under vacuum. Elemental analysis and mapping were carried out at the same location by Materials and Methods an EDS detector placed on the same optical axes of the SEM microscope. At all Sea Urchin Embryonic/Larval Culture. Ripe cultured adult sea urchins Para- stages, the sample was held at ambient conditions, with no exposure to the centrotus lividus were produced and supplied by the Israel Oceanographic vacuum environment. No additional processing was performed on the sample. and Limnological Research Institute. Spawning was induced by injecting 1 mL of 1 M KCl solution into the coelomic cavity. The egg suspension was collected, ACKNOWLEDGMENTS. We thank Vladimir Kiss and Reinat Nevo for their washed, and kept in sea water containing 30 mg/L penicillin (Sigma–Aldrich) help with confocal microscopy and Eugenia Klein, Elena Kartvelishvily, and and 15 mg/L streptomycin (Sigma–Aldrich) at 18 °C. Sperm was kept at 4 °C the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science for their help and guidance. The research undiluted. The fertilization was carried out up to 24 h after spawning by was supported by a German Research Foundation grant within the framework sperm dilution in sea water and rapid mixing with the egg suspension inside of the Deutsch–Israelische Projektkooperation and a Department of Energy sea water containing 30 mg/L penicillin and 15 mg/L streptomycin. The culture award (DE-FG02-07ER15899). L.A. is the incumbent of the Dorothy and Patrick was kept at 18 °C with gentle shaking (100 rpm). The developmental stage of Gorman Professorial Chair of Biological Ultrastructure, and S.W. is the incum- the embryo was determined using a light microscope with normal and bent of the Dr. Trude Burchardt Professorial Chair of Structural Biology.

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