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, 279 (1740), 3041-8

Biomineral Ultrastructure, Elemental Constitution and Genomic Analysis of Biomineralization-Related Proteins in Hemichordates

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Biomineral Ultrastructure, Elemental Constitution and Genomic Analysis of Biomineralization-Related Proteins in Hemichordates

C B Cameron et al. Proc Biol Sci.

Abstract

Here, we report the discovery and characterization of biominerals in the acorn worms Saccoglossus bromophenolosus and Ptychodera flava galapagos (Phylum: Hemichordata). Using electron microscopy, X-ray microprobe analyses and confocal Raman spectroscopy, we show that hemichordate biominerals are small CaCO(3) aragonitic elements restricted to specialized epidermal structures, and in S. bromophenolosus, are apparently secreted by sclerocytes. Investigation of urchin biomineralizing proteins in the translated genome and expressed sequence tag (EST) libraries of Saccoglossus kowalevskii indicates that three members of the urchin MSP-130 family, a carbonic anhydrase and a matrix metaloprotease are present and transcribed during the development of S. kowalevskii. The SM family of proteins is absent from the hemichordate genome. These results increase the number of phyla known to biomineralize and suggest that some of the gene-regulatory 'toolkit', if not mineralized tissue themselves, may have been present in the common ancestor to hemichordates and echinoderms.

Figures

Figure 1.
Figure 1.
(a) Light micrographs of S. bromophenolosus. (p, anterior proboscis; c, central collar; t, posterior trunk). (b) The trunk is spotted with numerous elevated white protuberances (pt, protuberances). (c) Four abutting protuberances located in the groove between the dorsolateral gonads and the ventral midline. Each protuberance is composed of several superficial and transparent spaces, some of which contain a single ossicle (s, spaces). Each of the four lines crosses a single protuberance at one of its narrower termini.
Figure 2.
Figure 2.
Scanning electron micrographs of ossicles isolated from S. bromophenolosus trunk epithelium. (a) Lateral view of a typical ossicle. The arrangement and shape of crystals differ between the central shaft and the termini. (b) Higher magnification of a terminus showing polycrystalline aggregates that form highly ordered, closely packed, expanding columns or trabeculae, of plate-like crystals (tb, trabeculae). (c) Higher magnification view showing the overlapping, smooth and laminar layers that constitute the central shaft portion of the ossicle in (a). (d) Terminal view of an ossicle, showing the arrangement of trabeculae, the perforated network of holes and a net-like matrix (arrows). (e) View of the inside of a fractured terminus. (f) View of the inside of a broken shaft.
Figure 3.
Figure 3.
Scanning electron micrographs of ossicles from the trunk epidermis of Ptychodera flava galapagos. (a) Ossicles are spherical with a mean diameter of 23.46 μm (±8.38, n = 20). (b) Ossicles are round and rugous. (c) They are composed of an outer layer that when fractured reveals a porous interior.
Figure 4.
Figure 4.
Transmission electron micrographs of sections through the epithelium of S. bromophenolosus showing the ossicles and sclerocyte cells. (a) The ossicles are positioned in a heavily ciliated epidermis (os, ossicles). (b) The border between an ossicle and its surrounding sheath of sclerocytes indicates an extracellular position. The cytoplasm is filled with transparent vesicles and abundant mitochondria (v, vesicles, mt, mitochondria). An electron-dense boundary layer (le, lamina externe) surrounds the cell body. (c) Sclerocyte cells form a sheath around the ossicle (sh, sheath). The inner sheath is lined with matrix material that presumably crosses the vacuolar space to be incorporated in the ossicle (vs, vacuolar space).
Figure 5.
Figure 5.
Determination of ossicle elemental constitution using X-ray microprobe analysis and crystal identity using confocal Raman spectroscopy. The ossicles of both species are composed of CaCO3, so only Saccoglossus data are shown. (a) Representative plot of EDS analysis of the central shaft of a single ossicle. (b) WDS analysis on 15 haphazardly chosen ossicles. Quantities are reported as mean weight % oxides. (c) Raman spectra for ossicles from S. bromophenolosus and a sea urchin test. The inset indicates the position on the hemichordate ossicle from which the spectral data were collected.

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