CRYSTAL GROWTH & DESIGN 2004 VOL. 4, NO. 4 667-669
Perspective Control of Crystal Growth in Biology: A Molecular Biological Approach Using Zebrafish Teresa Nicolson Oregon Hearing Research Center and Vollum Institute, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239 Received March 17, 2004;
Revised Manuscript Received April 29, 2004
ABSTRACT: This paper focuses on the in vivo manipulation of a gene encoding an acidic protein, Starmaker, which is critical for biomineralization of crystal structures in the zebrafish ear. Passive or active mineralization by animals is an ancient talent. Among vertebrates, the formation of the calcium phosphate skeleton is by far the best documented example of biomineralization. A very different, yet important, mineralized structure in our body is the otoconium, or “ear dust” formed in the inner ear. The crystals, which are an integral part of this structure, are composed of calcium carbonate. We use otoconia for our sense of balance. Like bones, these dense crystalline structures are prone to the effects of leaching over time, and defects in otoconia may account for many balance problems experienced by older people. Lower vertebrates form a variety of these crystal structures including single polycrystalline structures known as “ear stones” or otoliths (Figure 1). Higher vertebrate otoconia contain the polymorph calcite, the stablest form of calcium carbonate. The calcium carbonate polymorph in lower vetebrate inner ear crystals, especially those found in fish, are different. In the two larger otic chambers, aragonite is present, and in the smallest chamber, vaterite is always the polymorph formed. Why fish prefer these rarer polymorphs is still a mystery. Perhaps the denser forms of calcium carbonate increase the sensitivity of the sensory systems of the inner ear and are better suited to aquatic life. In zebrafish larvae, otoliths form on the first day of life. Initially, glycogen particles floating within the lumen of the developing ear cluster and attach to the first sensory hair cells.1,2 Fibrous organic matrix molecules attach to the cluster particles, presumably setting the stage for the first round of biomineralization. As the otolith grows, it loses its perfectly round shape and becomes more flattened like a thick sand dollar. The appearance of the otolith during the larval and adult stages is very glassy, as if made from pure crystal. At the electron microscopic level, rings of electron dense organic matter are apparent (Figure 1). Studies at the
Figure 1. Transmission electron micrograph of an otolithic organ in the zebrafish. The polycrystalline otolith is composed of aragonite crystals embedded in a fibrous organic matrix. In the ear, the otolith is coupled to a gelatinous membrane in contact with sensory hair cell bundles. Movements of the otolith in response to gravity and sound leads to shearing of sensory hair cell bundles. Upon deflection, cations flow into the sensory hair cells through mechanosensitive channels, causing depolarization. HB, hair bundle; HC, hair cell; OM, otolithic membrane; OT, otolith.
adult stage reveal that fish add a layer of calcium carbonate daily.3 This provides useful information to marine biologists since these rings indicate the age of the fish and whether its habitat has been conducive to growth.4 Studies of mineralized skeletal extracts from many vertebrate and invertebrate mineralizers have suggested that highly acidic proteins are important for biomineralization.5 Moreover, it is the protein component of the extract that determines which polymorph is formed. Single proteins capable of influencing polymorph selection have not been identified, mainly due to the difficulty of working with highly acidic proteins. They tend to aggregate and do not stain well on acrylamide gels. Several acidic proteins involved in bone formation have been identified, but a role in polymorph selection has not been established.6 One protein, dentin sialophosphoprotein (Dspp), is important for teeth for-
10.1021/cg0499010 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/17/2004
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mation in humans.7,8 Mutations in DSPP cause severe malformation of teeth. A large part of the Dspp protein includes stretches of aspartic acids and serines. Phosphorylated serine residues are also thought to be important for biomineralization.9 Thus, Dspp is an excellent candidate for direct participation in biomineralization of calcium phosphate mineral. Human geneticists who identified DSPP also noticed some cases of associated deafness with mutations in DSPP. Could the acidic Dspp protein be involved in mineralization in the same fashion in the ear, that is, is it also important for otoconia formation? Vestibular or balance defects were not noted in DSPP patients. Its role in the ear remains unclear. Starmaker protein is also very acidic like Dspp; however, it has additional features such as N-terminal repeats and its exon-intron structure is very different, suggesting that it is not an orthologue of Dspp.10 Starmaker appears to have a role in mineralization in zebrafish. Starmaker is present in the developing ear within 20 h after fertilization. At early stages, Starmaker is expressed at high levels in the epithelium during the phase when otolithic seeding particles are present in the lumen. Immunochemistry shows that Starmaker is secreted and binds to otolith precursor particles.10 Throughout all stages of otolith development, Starmaker is an integral component of otoliths. As development proceeds, expression of Starmaker is increasingly limited to just the sensory epithelium of the inner ear, the sites where otoliths are formed. Lying over the neuroepithelium, the developing otolith is exposed to more Starmaker protein at the surface nearest the neuroepithelium (Figure 1). Eventually, this surface of the otolith becomes flatter than the opposite side. Could the binding of Starmaker influence the shape or size of the crystals forming at this interface? Use of RNA antisense technology allows biologists to eliminate or radically decrease the levels of any protein by injecting antisense oligonucleotides into developing eggs or cells.11 Messenger RNA (mRNA) is normally translated into protein; however, in the presence of these antisense oligonucleotides, efficient translation or splicing is blocked. Antisense oligonucleotides (MO) directed against Starmaker RNA are very effective in removing the Starmaker protein.10 When the levels of Starmaker are decreased, aragonite crystals in otoliths become larger (Figure 2). In the complete absence of Starmaker, very large crystals form (Figure 2, 40 ng of MO). The shape of the otolith is also radically different depending on the levels of Starmaker protein. Wild-type otoliths have a round, smooth shape as described above. In the case of a moderate decrease in Starmaker levels, otoliths take on a starlike form, with apparent twinning events that are reminiscent of staurolite “fairy crosses” (Figure 2, 20 ng of MO). Indeed, in some cases, fairy cross-like otoliths were formed in animals injected with antisense oligonucleotides. In the majority of injected animals, however, double or triple twinning was more common. As the levels of Starmaker decrease, the flatness of the otolithic surface facing the neuroepithelium is also less prominent or lost altogether. Uncontrolled crystal growth upon loss of Starmaker activity suggests that Starmaker acts as a crystal inhibitor, regulating growth in a concentration-dependent man-
Perspective
Figure 2. Effects of decreased starmaker activity on otolith morphology and crystal lattice formation. Antisense oligonucleotides (MO) that interfere with splicing of starmaker mRNA were injected into zebrafish embryos, and otoliths were dissected out of 5 day-old larvae. Lower doses (4 ng) of MO cause irregularities of shape, although the size difference between anterior and posterior otoliths is preserved. Higher doses lead to increased crystal size (20 ng) and in the complete absence of Starmaker protein, a switch in crystal lattice from aragonite to calcite (40 ng).
ner. When Starmaker protein was completely absent, the otoliths were mainly composed of large inorganic crystals with very little organic fibrous matrix present (Figure 2, 40 ng of MO). The complex, rhombohedral faces of the crystals was suggestive of calcite, which was confirmed using X-ray microdiffraction experiments.10 The presence of calcite in the absence of Starmaker suggests that this polymorph is selected by default. It also suggests that Starmaker is required for the initial nucleation of aragonite. How does Starmaker select aragonite over calcite? The clue may lie in the spacing of the aspartic acid and serine residues. Earlier studies with peptides reveal that acidic residues or phosphorylated residues may provide a charged surface similar to the anionic component of calcium carbonate, thereby attracting calcium cations.9 If these residues are closely spaced, then a denser packing of ions may be possible. The effects of mutations in Dspp on hearing in humans have not been fully explored. Although no vestibular deficits have been reported in DSPP patients, it is possible that patients could compensate for a slight deficit. Examination of otoconia from DSPP patients would be insightful. On the other hand, it is worth noting that all sensory epithelia in the zebrafish ear express starmaker, including those in the semicircular canals.10 These cells are not associated with an otolithic structure, and no obvious defects are associated with these sensory organs in antisense oligonucleotideinjected animals. The role of Starmaker in these tissues is not clear. Expression in an additional tissue containing microcrystals, the pineal gland, is also evident in zebrafish.10 Further experiments may shed light on the various biological functions of Starmaker.
Perspective
The study of Starmaker and its role in the mineralization of the otoconia is one of the first, if not the first, example of using antisense oligonucleotides to probe the possible functions of a protein in crystal growth. As determining function is very difficult in these extracellular systems, this approach appears promising. To date, we know that many tens of proteins are present in mineralized tissues, but very few of their functions in controlling crystal growth have been identified, even in vitro. The ultimate hope is that when we know both protein structure and function, we will gain some deep insights from biology about how to control the complex process of crystal formation. Acknowledgment. I would like to thank Steve Weiner and Christian So¨llner for their helpful comments on the manuscript.
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