Otoc1: A novel otoconin‐90 ortholog required for otolith mineralization in zebrafish

Abstract

Within the vestibular system of virtually all vertebrate species, gravity and linear acceleration are detected via coupling of calcified masses to the cilia of mechanosensory hair cells. The mammalian ear contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while otolith matrix protein (OMP) is the most abundant matrix protein found in the otoliths of teleost fish. We have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 was detected in the ear between 15 hpf and 72 hpf, and was restricted primarily to the macula and the developing epithelial pillars of the semicircular canals. Expression of otoc1 was also detected in epiphysis, optic stalk, midbrain, diencephalon, flexural organ, and spinal cord. During embryogenesis, expression of otoc1 mRNA preceded the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produced a variety of aberrant otolith phenotypes. Our results suggest that Otoc1 may serve to nucleate calcium carbonate mineralization of aragonitic otoliths.

Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

“…The otolith abnormalities in our Sparc morphants encompassed all the main phenotypes of otoconin 90 (smaller, extra, or missing; Petko et al 2008), otolith matrix protein (smaller; Murayama et al 2005), and otolin-1 (fused; Murayama et al 2005), but appeared to be opposite to that of starmaker (reduced vs. enhanced mineralization; Söllner et al 2003). This would be consistent with, but not proof of genetic or physical interactions between Sparc and some or all of these otolith proteins.…”

“…Previously, constituent otolith proteins had been isolated one at a time by electrophoresis (Murayama et al 2000(Murayama et al , 2002, database searches (Söllner et al 2003;Petko et al 2008), or immunoscreening (Tohse et al 2008). Recently, Zhao et al (2007) employed mass spectrometry to detect three proteins in a crude extract from mouse otoconia: otoconin 90 (Wang et al 1998;Verpy et al 1999) and fetuin-A as well as otolin-1, whose ortholog had previously been isolated from salmon otoliths (Murayama et al 2000).…”

“…Only five proteins have been located in otoliths or otoconia as well as functionally characterized. First, otoconin 90, the major otoconia protein, in mouse (Wang et al 1998;Verpy et al 1999;Zhao et al 2007) and zebrafish (Petko et al 2008), and its homolog otoconin 22 in frog (Pote et al 1993;Yaoi et al 2004). Second, otolith matrix protein-1, the major otolith protein, in trout and zebrafish (Murayama et al 2000(Murayama et al , 2004(Murayama et al , 2005.…”

Proteins Involved in Otoconia Formation and Maintenance

“…Oc90 expression is the earliest among all otolith/otoconia proteins in fish and mice (before embryonic day E9.5 in mice) (Petko et al 2008;Verpy et al 1999;Wang et al 1998), much earlier than the onset of any activities of ion channels/pumps, or the onset of otoconia seeding at around E14.5. Oc90 then recruits other components at the time of their expression to form the organic matrix for calcification (Zhao et al 2007).…”

“…When otoconia growth stops at around P7 (postnatal day 7), the expression level of Oc90 significantly decreases in the utricle and saccule . Although Oc90 has a relatively low abundance in zebrafish otoliths (known as zOtoc1) (Petko et al 2008), Oc90 morphant fish show more severe phenotypes than morphants for the main otolith matrix protein OMP1 (Murayama et al 2005;Petko et al 2008), suggesting that zOc90 (zOtoc1) is essential for the early stages of otolith development (i.e. crystal seeding) whereas OMP regulates crystal growth.…”