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Thiessen KD
,
Grzegorski SJ
,
Chin Y
,
Higuchi LN
,
Wilkinson CJ
,
Shavit JA
,
Kramer KL
.
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.
Figure 2:. Complementary approaches for causative gene discovery. MMAPPR analysis of RNA sequencing data for nco (A) and whole genome homology mapping for csr (B) identified regions of high homology on the 24th chormosome near the pks1 locus (~33 Gb). (C) Deleterious mutations were identified in pks1 for nco and csr within the acyl transferase (AT) domain and vns within the polyketide synthase (PKS) domain. Sanger sequencing confirmed SNPs in csr, nco, and vns mutants. Other domains include Ketoacyl Synthetase (KS), Medium Chain Reductase (MDR), NAD(P)-dependent dehydrogenase (NDD), and Phosphopanthetheine-Binding (PP).
Figure 3:. WT pks1 nucleic acid rescues otolith formation in csr, nco, and vns. (A) Normal frequencies of mutant phenotypes in each uninjected strain. All four pairings follow homozygous recessive mode of inheritance. (B) Results of injected embryos show that Japanese medaka pks1 mRNA (300 pg) rescues both csr and nco mutants and pks1 DNA (20 pg) rescues vns mutants. (*, p < 0.0001, paired t-test)(**, p < 0.0032, paired t-test)(***, p = 0.0001, paired t-test),. Site-directed mutagenesis was used to introduce a conserved mutation in csr (A911P) into the Japanese medaka construct (L905P) (C) Injection of pks1L905P (300 pg) fails to rescue csr or nco mutant phenotypes.
Figure 4:. Gene expression and pathway analysis of nco embryos. (A) Ingenuity Pathway Analysis shows the top up-regulated and down-regulated pathways, which are eNOS Signaling and Endothelin-1 Signaling, respectively. Positive z-score indicated increased mRNA levels. Negative z-score indicates decreased mRNA levels. No change in mRNA levels results in a z-score of zero. (B) Differential gene expression in the top up-regulated genes. (C) Differential gene expression in the top down-regulated genes. (**, expressed in adult zebrafish mechanosensory hair cells) [32].
Figure 5:. Aberrant expression of proteins invovled in otolith development in csr and nco. (A) Schematic of anterior macula (AM) tethered to otolith at 27 hpf. (B) In WT, Otoconin-90 (Oc90) is expressed within the mineralized otolith, which is situated atop the otolithic membrane (Otogelin, or Otog), at 27 hpf. Scale bar = 5 μm. (C-D) Oc90 has diffuse expression within the otocyst of csr and nco. In csr and nco, Otog is localized near the apical surface of hair cells. (E-F) Expression showing hair cells in WT and nco larvae at 5dpf. Scale bar = 25 μm. (G) Quantification of hair cell numbers in the posterior and anterior macula of WT and nco (n = 4).
