Polyketide Synthase is Required for Zebrafish Otolith Biomineralization
Authors: Thiessen Kevin , Higuchi Lisa , Kramer Kenneth 
Published: November 2023

ObjectiveDeflecting biomineralized crystals attached to vestibular hair cells is necessary for maintaining the body's 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 pair of genetically‐linked mutants, no content (nco) and corkscrew (csr), that fail to develop otoliths during early ear development.MethodsMutant nco embryos and wild‐type clutchmates (WT) were collected during the critical period of otolith nucleation (24 hours post fertilization, hpf) and submitted for RNA sequencing. Analysis was completed using MMAPPR (Mutation Mapping Analysis Pipeline for Pooled RNA‐seq). Whole genome sequencing of csr was performed and analyzed using MegaMapper. WT mRNA was synthesized using mMessage Machine from a clone provided by Dr. Hiroyuki Takeda (University of Tokyo), cleaned on an RNeasy column, and subsequently injected into single‐cell csr and nco embryos. For immunofluorescence (IF), csr embryos were collected during key stages in ear development, fixed with hydrogel and washed in CLARITY‐clearing solution. Embryos were decalcified with EDTA before blocking, incubating in primary and secondary antibodies, and imaging by confocal microscopy. To test the effects of exogenous calcium ions on otolith formation, embryos were kept in E3 Medium until early gastrulation. Embryos were washed and transferred to 1X Basic Solution (58 mM NaCl, 0.4 mM MgSO4 and 5 mM HEPES) supplemented with 0.7 mM potassium chloride [0 mM Ca2+], 0.6 mM calcium nitrate [0.15 mM Ca2+] or 0.6 mM calcium chloride [0.22 mM Ca2+]. Embryos were scored by the presence or absence of otoliths at 27 hpf. Statistical significance was calculated using Fisher's Exact Test and Chi‐Squared Distribution or by Linear Regression.ResultsTo positionally clone the gene responsible for nco and csr, we used complementary approaches for each strain. MMAPPR analysis of nco‐derived RNA sequencing and MegaMapper analysis of csr‐derived whole genome sequencing both identified a genomic region surrounding polyketide synthase 1 (pks1; current gene name wu:fc01d11), a candidate gene that was recently identified as a key factor of otolith biomineralization in Japanese medaka (Oryzias latipes, Ol). Microinjection of OI pks1 mRNA rescued otolith biomineralization in both csr (p<0.0001; χ2<0.0001) and nco (p=0.0032; χ2=0.0022) mutants. Using IF, an otoconial seeding protein, Otoconin‐90, and an otolith matrix protein, Starmaker, show diffuse expression inside the otocyst of csr mutant embryos. Expression of Otogelin, a primary component of the otolithic membrane, is localized near the apical surfaces of sensory hair cells in both csr and WT. Additionally, exogenous calcium concentrations correlate with penetrance of otolith formation in csr embryos (R2=0.9978); however, nco embryos were unaffected (R2=0.7802).ConclusionsOtolith nucleation is impaired in the genetically‐linked zebrafish mutants csr and nco. IF of csr embryos demonstrated that expression of a critical otoconial seeding protein, Otoconin‐90, within the otocyst is not sufficient for otolith biomineralization in the presence of the otolithic membrane. While WT Ol pks1 rescues otolith biomineralization in csr and nco, differences in penetrance of calcium ions on otolith formation suggest the nature of each mutation is fundamentally different.Support or Funding InformationThe work was supported by grants from the National Institutes of Health (8P20GM103471) and the Creighton Health Science Strategic Investment Fund.