Otolith Defects Research Articles

Radial spoke proteins regulate otolith formation during early zebrafish development.

Radial spokes are structurally conserved, macromolecular complexes that are essential for the motility of 9 + 2 motile cilia. In Chlamydomonas species, mutations in radial spoke proteins result in ciliary motility defects. However, little is known about the function of radial spoke proteins during embryonic development. Here, we investigated the role of a novel radial spoke protein, leucine-rich repeat containing protein 23 (Lrrc23), during zebrafish embryonic development. Mutations in lrrc23 resulted in a selective otolith formation defect during early ear development. Similar otolith defects were also present in the radial spoke head 3 homolog ( rsph3) and radial spoke head 4 homolog A ( rsph4a) radial spoke mutants. Notably, the radial spoke protein mutations specifically affected ciliary motility in the otic vesicle (OV), whereas motile cilia in other organs functioned normally. Via high-speed video microscopy, we found that motile cilia formation was stochastic and transient in the OV. Importantly, all the motile cilia in the OV beat circularly, in contrast to the planar beating pattern of typical 9 + 2 motile cilia. We identified the key time frame for motile cilia formation during OV development. Finally, we showed that the functions of radial spoke proteins were conserved between zebrafish and Tetrahymena. Together, our results suggest that radial spoke proteins are essential for ciliary motility in the OV and that radial spoke-regulated OV motile cilia represent a unique type of cilia during early zebrafish embryonic development.-Han, X., Xie, H., Wang, Y., Zhao, C. Radial spoke proteins regulate otolith formation during early zebrafish development.

A founder CEP120 mutation in Jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies.

Jeune asphyxiating thoracic dystrophy (JATD) is a skeletal dysplasia characterized by a small thoracic cage and a range of skeletal and extra-skeletal anomalies. JATD is genetically heterogeneous with at least nine genes identified, all encoding ciliary proteins, hence the classification of JATD as a skeletal ciliopathy. Consistent with the observation that the heterogeneous molecular basis of JATD has not been fully determined yet, we have identified two consanguineous Saudi families segregating JATD who share a single identical ancestral homozygous haplotype among the affected members. Whole-exome sequencing revealed a single novel variant within the disease haplotype in CEP120, which encodes a core centriolar protein. Subsequent targeted sequencing of CEP120 in Saudi and European JATD cohorts identified two additional families with the same missense mutation. Combining the four families in linkage analysis confirmed a significant genome-wide linkage signal at the CEP120 locus. This missense change alters a highly conserved amino acid within CEP120 (p.Ala199Pro). In addition, we show marked reduction of cilia and abnormal number of centrioles in fibroblasts from one affected individual. Inhibition of the CEP120 ortholog in zebrafish produced pleiotropic phenotypes characteristic of cilia defects including abnormal body curvature, hydrocephalus, otolith defects and abnormal renal, head and craniofacial development. We also demonstrate that in CEP120 morphants, cilia are shortened in the neural tube and disorganized in the pronephros. These results are consistent with aberrant CEP120 being implicated in the pathogenesis of JATD and expand the role of centriolar proteins in skeletal ciliopathies.

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

A novel role for the obesity-associated gene FTO in ciliogenesis and Wnt signalling

Common intronic variants in hFTO, an oxoglutarate-dependent dioxygenase, were identified in GWAS to cause a predisposition to obesity. Inactivation of Fto in human and mouse models results in reduced viability, postnatal growth retardation, enhanced metabolism and loss of adipose tissue. However, the function of Fto during development and obesity remains unclear. We sought to clarify the function of Fto using zebrafish and cell culture. In zebrafish, we found loss of fto results in a ciliopathy-like phenotype. Morphants display small eyes, curved tails, otolith defects, disorganised melanocytes, situs inversus and renal cysts. Concurrently, cilia were absent in the olfactory bulbs and disorganised in the pronephric ducts (PND) and neural tube. Functional analysis of motile cilia in the PND and Kupffer’s vesicle confirmed a direct ciliary defect. Wnt signalling has been implicated in adipogenesis and therefore we decided to investigate whether Fto may interact at this level. We found that β-Catenin and downstream target genes are consistently downregulated in fto morphants, HEK293T cells transfected with an shRNA Fto expressing plasmid, and Fto-/- MEFs. Furthermore, β-Catenin failed to translocate to the nucleus in Fto-/- MEFs in response to Wnt3a stimulation. By completing a comparative Wnt signalling phospho-antibody array, on Fto+/+ and Fto-/- MEFs, we identified increased PKC and CamKII activity: two non-canonical/Ca2+ Wnt components. Indeed, phospo-CamkII is up regulated in fto morphants and in response to Wnt3a in Fto-/- MEFs. Taken together, these data show that Fto acts as a key cilia-mediated regulator of canonical and non-canonical/Ca2+ Wnt signalling.

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC 50) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.

Developmental toxicity of dinocap in the mouse is not due to two isomers of the major active ingredients.

Technical-grade dinocap, a complex-mixture fungicide, is teratogenic in the CD-1 mouse, causing cleft palate and otolith defects. In this study we compared the developmental toxicities of 2,4-dinitro-6-(1-methylheptyl)phenyl crotonate and 2,6-dinitro-4-(1-methylheptyl)phenyl crotonate, model isomers of the major active ingredients of technical dinocap, to the known teratogenicity of the technical compound. Individual isomers, both isomers combined, or technical dinocap were administered to pregnant mice on days 7-16 of gestation. Some dams were killed at term and litters were removed, dead fetuses and resorptions were counted, and live fetuses were weighed and preserved in Bodian's fixative for examination for cleft palate. Other treated dams were allowed to give birth: postnatal viability and growth, development of swimming behavior, and otolith formation were evaluated. As in previous studies, technical-grade dinocap caused cleft palate and weight deficits in fetuses at term and increased neonatal mortality and abnormal swimming behavior, torticollis, and deficient otolith formation in surviving pups. Neither of the purified isomers exhibited any developmental toxicity when administered under identical conditions. Thus, it is concluded that these isomers are not the active teratogenic component(s) in technical-grade dinocap.

Zinc metabolism in lethal-milk mice. Otolith, lactation, and aging effects.

Lethal-milk (lm), a recessive mutation, occurred in the C57BL/6J inbred strain of mice. Lactating lm dams produce a zinc-deficient milk that is lethal to all nursing pups. If foster-nursed on normal dams, lm pups survive and become reproductively mature. Injection of zinc-glycinate into the pups or zinc supplementation of the water of the lactating dams reduces lethality. Other pleiotropic effects in lm mice include congenital otolith defects with delayed righting, "tail-spinning," and abnormal swimming. These effects are diagnostic criteria for segregation of lm mice among backcross progeny. About 40 percent of the expected number of lm pups survive to weaning. Zinc supplementation of the dam improves development of saccular but not of utricular otoliths; zinc does not improve survival of the lm pups among backcross progenies. The lm mice over eight months of age also exhibit extensive hair loss, dermatitis, and skin lesions. Possible roles of metallothionein in zinc and copper metabolism are discussed in regard to the pleiotropic effects of the lethal-milk mutation.

Trace metals and otolith defects in mocha mice

Mocha mice with pigment anomalies of the coat, eyes, and inner ears also have congenital otolith defects, and they exhibit progressive cochlear degeneration. Mocha mice were first reported to exhibit otolith defects comparable to those of pallid mice. Since manganese supplementation is effective in preventing the otolith defects in pallid mice and in pastel mink, we sought to establish whether or not manganese also might be effective in mocha mice. The otolith defects of mocha mice were prevented or reduced by supplementing the pregnant dams with manganese and/or zinc. The mocha mice also exhibited high perinatal mortality that was not reduced by the supplementary metals. Surviving mocha mice have behavioral anomalies associated with their inner ear defects. Preliminary observations of auditory-evoked brainstem responses and of cochlear degeneration in the mocha mice are discussed.

Influence of manganese on genetically defective otolith. A behavioral and morphological study.

The genetically mutant pallid mouse (which possesses specific otolith defects) and the manganese-supplemented (Mn) pallid mouse (in which otolith defects were prevented) were studied using light and transmission and scanning electronmicroscopy. Untreated pallid mice which showed head-tilting, circling and ataxia also had swimming difficulty. In these animals, the otolith defects were the only consistent findings. With one exception, no pronounced anomalies were found in the sensory epithelium, nerve fibers and ganglion cells. Only a few vestibular melanocytes, in which the melanin granules failed to fully mature, were observed in the pallid mouse. Melanization in vestibular melanocytes appeared to be improved by Mn-supplementation. The genetic basis for involvement of Mn in otolith development was discussed. This animal model provides opportunities for basic research concerning the possible prevention of genetic disorders involving the inner ear.

Effects of dichlorophenamide, zinc, and manganese on otolith development in mice

Evidence is presented suggesting that carbonic anhydrase and zinc affect the deposition of otoconia, or calcium carbonate crystals, during otolith development in mice. A single injection of dichlorophenamide (an inhibitor of carbonic anhydrase) into pregnant Swiss/COX albino females at a specific day of gestation (13–18) produced otolith defects in the progeny as observed shortly after birth. Similar defects were found in progeny of pregnant females fed zinc-deficient diets beginning at various days of gestation (0, 10–16). Despite the highly significant effects of the drug and of the zinc-deficient diet, untreated controls exhibited a low incidence (approximately 15%) of otolith defects. Various purified diets were employed to measure the effects of varying ratios of manganese and zinc on otoliths. Diets fortified with manganese plus zinc completely eliminated otolith defects. All other diets employed, significantly reduced the incidence of defects. It is postulated that: (1) the most active rate of crystal formation occurs during days 15 and 16 of gestation; (2) an observed asymmetry may reflect differences in the rate of development of ear primordia, or in levels of carbonic anhydrase activity; (3) the otoconia may undergo a rapid metabolic turnover, or they may be affected by subtle changes in pH of the endolymph; and (4) manganese and zinc are essential for normal otolith development.

Congenital ataxia and otolith defects due to manganese deficiency in mice.

Manganese deficiency in mice caused congenital ataxia in some prog eny. The most diagnostic means of detecting the ataxia was to place all mice in a tank of water and observe their ability to maintain a normal, upright position. The incidence of ataxia increased approximately in proportion to the duration and severity of the manganese-deficient state of the mother. Cleared otic capsules revealed that otoliths were reduced in size, or were absent, in many of the manganese-deficient progeny. Supplementation of the manganese-deficient females after day 14 of gestation was ineffective in reducing the incidence of the otolithic effect. Supplementation between days 12 and 14 was only partially effective, whereas supplementation starting on day 11 was sufficient for completely normal development of otoliths. The absence of oto lithic crystals is attributed to a defect in the synthesis of the organic matrix which appears to be composed of acid mucopolysaccharides. The absence of otoliths was also found in manganese-deficient chicks and presumably explains the long-standing reports of ataxia due to manganese deficiency in poultry and other domestic animals.