Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

Large-scale mouse mutagenesis initiatives have provided new mouse mutants that are useful models of human deafness and vestibular dysfunction. Catweasel is a novel N-ethyl-N-nitrosourea (ENU)-induced mutation. Heterozygous catweasel mutant mice exhibit mild headtossing associated with a posterior crista defect. We mapped the catweasel mutation to a critical region of 13 Mb on chromosome 12 containing the Six1, -4 and -6 genes. We identified a basepair substitution in exon 1 of the Six1 gene that changes a conserved glutamic acid (E) at position 121 to a glycine (G) in the Six1 homeodomain. Cwe/Cwe animals lack Preyer and righting reflexes, display severe headshaking and have severely truncated cochlea and semicircular canals. Cwe/Cwe animals had very few hair cells in the utricle, but their ampullae and cochlea were devoid of any hair cells. Bmp4, Jag1 and Sox2 expression were largely absent at early stages of sensory development and NeuroD expression was reduced in the developing vestibulo-acoustic ganglion. Lastly we show that Six1 genetically interacts with Jag1. We propose that the catweasel phenotype is due to a hypomorphic mutation in Six1 and that catweasel mice are a suitable model for branchio-oto-renal syndrome. In addition Six1 has a pivotal role in early sensory patch development and may act in the same genetic pathway as Jag1.

The caudal fin of adult zebrafish is used to study the molecular mechanisms that govern regeneration processes. Most reports of gene expression in regenerating caudal fins rely on in situ hybridization (ISH) on whole-mount samples followed by sectioning of the samples. In such reports, expression is mostly confined to cells other than those located between the dense collagenous structures that are the actinotrichia and lepidotrichia. Here, we re-examined the expression of genes by performing ISH directly on cryo-sections of regenerates. We detected expression of some of these genes in cell types that appeared to be non-expressing when ISH was performed on whole-mount samples. These results demonstrate that ISH reagents have a limited capacity to penetrate between the regenerating skeletal matrices and suggest that ISH performed directly on fin sections is a preferable method to study gene expression in fin regenerates.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA2, 3. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

Photoreceptor cell degeneration was not detected in any of the shaker1 alleles, except for a small but significant loss of photoreceptor cells found in Myo7a(4626SB/4626SB) mice that were also homozygous mutant for Cdh23v. Perhaps greater and/or faster photoreceptor cell loss that is dependent on mutant Myo7a can be effected by having additional mutant USH1 genes in the genetic background. In any case, it is argued that shaker1 mice are a useful model for testing USH1B gene therapy, due to the presence of mutant phenotypes other than photoreceptor cell death.

The analysis of mouse mutants has become an almost indispensable tool in modern biology. The understanding of inner ear development has been facilitated by the use of mice from chemical- and radiation-induced mutagenesis screens, as well as gene-targeting strategies such as knockout models. In particular, our understanding of hereditary gross malformations and neuroepithelial defects that underlie human deafness has been aided by the analysis of comparable gene defects in mice. Mice with mutations in transcription factor genes such as Pax2 and Hmx3 have illustrated the early compartmentalization of the otocyst, whereas defects in endolymph homeostasis, such as those found in Pendred syndrome, show how malformations of the inner ear can arise through fluid changes within it. In addition, a surprisingly large number of mice with hearing and balance problems exhibit defects in sensory hair cell differentiation. From these animals we have been able to identify key gene pathways, such as the Notch signaling pathway, that are involved in cell fate determination and maturation in the inner ear. We have also been able to identify some of the major structural, cytoskeletal components of the mechanotransducing hair cells, such as unconventional myosins and other motor proteins such as prestin. Analysis of mutant mice has been incredibly useful in the identification of functionally, and therefore clinically, important genes and together with recent molecular advances, such as the sequencing of the mouse genome, have taken mouse genetics to a new level that will surely make it indispensable in the future analysis of mammalian development.

Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. We established an in vivo method of gene transfection to express ectopically genes in the blastema of regenerating fins. Ectopic expression of shh or bmp2 in the blastema-induced excess bone deposition and altered patterning of the regenerate. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of shh expression and a rapid abolition of ptc1 expression. These results implicate shh and bmp2b signaling in the proliferation and/or differentiation of specialized bone-secreting cells in the blastema and suggest shh expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.

Chemokines, in addition to their characterized functions as immune modulators, also play a role in developmental processes such as neural cell migration. Although, chemokines have been described in human, mouse and other vertebrate species, they have yet to be characterized in zebrafish. In this paper, we report the isolation and expression analysis of scyba, a zebrafish gene encoding a CXC-type chemokine protein. During early segmentation, scyba transcripts are detected in the midbrain region and the otic placodes. At later developmental stages, scyba expression is restricted to a subset of hindbrain commissural neurons and to the hair-cell sensory patches of the otic vesicle and lateral-line neuromasts.

Dlx homeobox genes of vertebrates are often organised as physically linked pairs in which the two genes are transcribed convergently (tail-to-tail arrangement). Three such Dlx pairs have been found in mouse, human, and zebrafish and are thought to have originated from the duplication of an ancestral gene pair. These pairs include Dlx1/Dlx2, Dlx7/Dlx3, and Dlx6/Dlx5 (the zebrafish orthologue of Dlx5 is named dlx4). Expression patterns of physically linked Dlx genes overlap extensively. Furthermore, orthologous Dlx genes often show highly similar expression patterns. We analysed Dlx expression during the gastrula and early somitogenesis of the mouse and zebrafish. It was found that expression of the mouse Dlx6 gene takes place in the rostral ectoderm and presumptive olfactory and otic placodes with patterns similar to the previously reported expression of the physically linked Dlx5 gene. However, we observed only very weak expression of the mouse Dlx3 gene at the same stage. This contrasts with the expression of dlx genes in zebrafish where dlx3 and dlx7, but not dlx4 and dlx6 are expressed during gastrulation in the rostral ectoderm and presumptive placodes. Thus, Dlx expression patterns at early stages are better conserved between paralogous pairs of physically linked genes than between orthologous pairs. This suggests that early expression of Dlx genes existed prior to the duplications that led to the multiple pairs of physically linked genes but was differentially conserved in different paralogs in zebrafish and mice.

Recent reports suggest that immature hair bundles are observed following aminoglycoside-induced hair-cell loss in the mammalian utricle in vitro as well as in vivo. It is therefore important to document the initial morphological changes associated with both culturing and aminoglycoside application so that degeneration can be clearly distinguished from regeneration. In this study, utricles from adult guinea pigs were maintained in culture for either 3 or 8 days, half being exposed to neomycin for days 2 and 3. They were then processed for microscopical examination and compared with control utricles from animals of the same age. The numbers of hair-cell and supporting-cell nuclei were counted and hair-cell morphology assessed. Bundles were classified as having either stepped (SHB) or unstepped (UHB) stereocilia, and their density determined. The numbers of hair-cell, but not supporting-cell, nuclei declined significantly compared with controls in both untreated and treated explants, the greatest reduction occurring 5 days after neomycin administration. The density of SHBs also declined but there was no significant change in UHB density, resulting in a residual population of hair bundles of more immature appearance in both untreated and treated utricles in vitro than in vivo. Although degenerative events such as hair-cell ejection from, or retraction into, the sensory epithelium were observed, no evidence of regeneration was found.

Annals of the New York Academy of SciencesVolume 781, Issue 1 p. 683-685 The Effect of Neomycin on Organotypic Cultures of the Adult Guinea-Pig Utriclea E. QUINT, E. QUINT Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this authorC. M. HACKNEY, C. M. HACKNEY Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this authorD. N. FURNESS, D. N. FURNESS Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this author E. QUINT, E. QUINT Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this authorC. M. HACKNEY, C. M. HACKNEY Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this authorD. N. FURNESS, D. N. FURNESS Department of Communication and Neuroscience Keele University Keele, Staffordshire ST5 5BG United KingdomSearch for more papers by this author First published: June 1996 https://doi.org/10.1111/j.1749-6632.1996.tb15759.xCitations: 1 a This work was supported by the BBSRC, Hearing Research Trust, and Wellcome Trust. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume781, Issue1New Directions in Vestibular ResearchJune 1996Pages 683-685 RelatedInformation