Zebrafish otolith biomineralization requires polyketide synthase

Abstract

Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish ( Danio rerio ) are useful organisms to study these biomineralized crystals called otoliths, as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a trio of homozygous recessive mutants, no content ( nco ) and corkscrew ( csr ), and vanished ( vns ), which fail to develop otoliths during early ear development. We show that nco , csr , and vns have potentially deleterious mutations in polyketide synthase ( pks1 ), a multi-modular protein that has been previously implicated in biomineralization events in chordates and echinoderms. We found that Otoconin-90 (Oc90) expression within the otocyst is diffuse in nco and csr ; therefore, it is not sufficient for otolith biomineralization in zebrafish. Similarly, normal localization of Otogelin, a protein required for otolith tethering in the otolithic membrane, is not sufficient for Oc90 attachment. Furthermore, eNOS signaling and Endothelin-1 signaling were the most up- and down-regulated pathways during otolith agenesis in nco , respectively. Our results demonstrate distinct processes for otolith nucleation and biomineralization in vertebrates and will be a starting point for models that are independent of Oc90-mediated seeding. This study will serve as a basis for investigating the role of eNOS signaling and Endothelin-1 signaling during otolith formation.

The biosynthetic diversity of the animal world

“…Another PKS characterized by genetic means, but with uncertain products, is OlPKS found in the zebrafish. Nonsynonymous mutation in the OlPKS KS domain led to deficiencies in otolith (hearing) formation in the fish and is related to calcium accumulation (36). This function is conceptually similar JBC REVIEWS: The biosynthetic diversity of the animal world to the role of Pks2 result from urchins.…”

“…In the course of these studies, the investigators identified many other PKSs in animals of many different phyla, showing that animal PKSs are widespread (33,34,36,38). Intriguingly, whereas the domain order of the PKS is somewhat conserved at the protein N terminus, the C-terminal ends of animal PKSs are highly variable, containing different termination domains, and even in several cases NRPS domains.…”

Cilia in the developing zebrafish ear

“…Otolith precursor particles are initially found distributed throughout the otic vesicle lumen, but adhere specifically to the tips of kinocilia on the first hair cells to differentiate in the ear (named tether cells for this reason) [2,3,9]. The composition of the particles has not been fully described, but they are likely to contain Cadherin11 [40], Otoconin90 [41,42] and Starmaker [43,44]. At the kinociliary tip, the bound otolith precursor particles serve as a nucleation site for deposition of further layers of protein and mineral (calcium carbonate), and thus the tether kinocilia become partially embedded within the structure of the otolith.…”

“…At the kinociliary tip, the bound otolith precursor particles serve as a nucleation site for deposition of further layers of protein and mineral (calcium carbonate), and thus the tether kinocilia become partially embedded within the structure of the otolith. Efficient production or nucleation of the precursor particles, in both zebrafish and medaka, appears to require the activity of the enzyme polyketide synthase [41,45,46]. Growing zebrafish otoliths incorporate a number of proteins, including Otolith Matrix Protein-1, Sparc, Starmaker and others [47][48][49][50].…”

“…Otogelin remains the best candidate to date for an otolith precursor-binding factor. Although expression of otogelin mRNA is not restricted to hair cells [66], staining with an anti-Otogelin antibody suggests that the protein itself becomes concentrated at the distal half of the tether kinocilia [41]. How Otogelin gets there is not currently understood.…”