Zebrafish pax5 regulates development of the utricular macula and vestibular function.

The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.     

Scanning electron microscopic study of the sacculus and lagena in several deep-sea fishes

The ultrastructure of the sacculus and lagena, two otolithic organs involved in audition, was studied in seven species of bathypelagic and mesopelagic fishes representing a taxonomically diverse sampling of Division-III teleost fishes. The saccular macula in each species had hair cells oriented in four directions, with cells on the rostral part of the macula oriented anteriorly and posteriorly, and those on the caudal end of the macula oriented dorsally and ventrally. The most significant variation from this pattern was in a Gadiform fish, Bregmaceros sp., which also had additional groups of horizontally oriented cells on the posterior end of the macula. The lagenar macula in each species had very similar hair cell orientation patterns, there being dorsally oriented cells on the anterior side of the macula and ventrally oriented cells on the posterior side. The only exception to this pattern was in Ectreposebastes, in which the two groups of cells were oriented towards one another. Significantly, the predominant ciliary bundles on the sensory hair cells of the saccular maculae, and to a slightly lesser degree on the lagenar maculae, were quite similar in almost all species. The bundles had a single long kinocilium and graded stereocilia, the longest of which were almost as long as the kinocilium. This pattern is far less frequently found in shallow water fishes. These data further demonstrate that the hair cells oriented in four directions on the saccular macula may be ubiquitous among all teleost fishes other than the Ostariophysi. The data also lead to the suggestion that the elongate ciliary bundle may be adaptive to certain features of life in deep water.     

New role of LRP5, associated with nonsyndromic autosomal‐recessive hereditary hearing loss

Human hearing loss is a common neurosensory disorder about which many basic research and clinically relevant questions are unresolved. At least 50% of hearing loss are due to a genetic etiology. Although hundreds of genes have been reported, there are still hundreds of related deafness genes to be found. Clinical, genetic, and functional investigations were performed to identify the causative mutation in a distinctive Chinese family with postlingual nonsyndromic sensorineural hearing loss. Whole‐exome sequencing (WES) identified lipoprotein receptor‐related protein 5 (LRP5), a member of the low‐density lipoprotein receptor family, as the causative gene in this family. In the zebrafish model, lrp5 downregulation using morpholinos led to significant abnormalities in zebrafish inner ear and lateral line neuromasts and contributed, to some extent, to disabilities in hearing and balance. Rescue experiments showed that LRP5 mutation is associated with hearing loss. Knocking down lrp5 in zebrafish results in reduced expression of several genes linked to Wnt signaling pathway and decreased cell proliferation when compared with those in wild‐type zebrafish. In conclusion, the LRP5 mutation influences cell proliferation through the Wnt signaling pathway, thereby reducing the number of supporting cells and hair cells and leading to nonsyndromic hearing loss in this Chinese family.     

A novel PLS1 c.981+1G>A variant causes autosomal-dominant hereditary hearing loss in a family

The fimbrin protein family contains a variety of proteins, among which Plastin1 (PLS1) is an important member. According to recent studies, variations in the coding region of the PLS1 gene are associated with the development of deafness. However, the molecular mechanism of deafness caused by PLS1 gene variants remains unknown. Whole-exome sequencing was performed on hearing-impaired family members and hearing family members to identify pathogenic variants, followed by Sanger sequencing. A minigene assay was conducted to investigate the effect of the variant on PLS1 mRNA splicing. The pathogenicity of the variant was further investigated in zebrafish. RNA-sequencing (RNA-seq) was performed to analyze the dysregulation of downstream signaling pathways caused by knockdown of PLS1 expression. We identified a novel variant, PLS1 c.981+1G>A, in a large Chinese family with hearing loss and showed that the variant is responsible for the occurrence of hearing loss by inducing exon 8 skipping. The variant caused abnormal inner ear phenotypes, characterized by decreases in the mean otolith distance, anterior otolith diameter, posterior otolith diameter, cochlear diameter, and swimming speed and distance in zebrafish. Furthermore, silencing PLS1 expression significantly upregulated the expression of genes in the PI3K-Akt signaling pathway, including Col6a3, Spp1, Itgb3 and hepatocyte growth factor (Hgf). PLS1 c.981+1G>A is a novel pathogenic variant causing hearing loss by inducing exon 8 skipping. Upregulation of the expression of genes in the PI3K-Akt signaling pathway plays an important role in the pathogenesis caused by variants in the PLS1 gene.     

Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness

The zebrafish (Danio rerio) possesses two mechanosensory organs believed to be homologous to each other: the inner ear, which is responsible for the senses of audition and equilibrium, and the lateral line organ, which is involved in the detection of water movements. Eight zebrafish circler or auditory/vestibular mutants appear to have defects specific to sensory hair cell function. The circler genes may therefore encode components of the mechanotransduction apparatus and/or be the orthologous counterparts of the genes underlying human hereditary deafness. In this report, we show that the phenotype of the circler mutant, mariner, is due to mutations in the gene encoding Myosin VIIA, an unconventional myosin which is expressed in sensory hair cells and is responsible for various types of hearing disorder in humans, namely Usher 1B syndrome, DFNB2 and DFNA11. Our analysis of the fine structure of hair bundles in the mariner mutants suggests that a missense mutation within the C-terminal FERM domain of the tail of Myosin VIIA has the potential to dissociate the two different functions of the protein in hair bundle integrity and apical endocytosis. Notably, mariner sensory hair cells display morphological and functional defects that are similar to those present in mouse shaker-1 hair cells which are defective in Myosin VIIA. Thus, this study demonstrates the striking conservation of the function of Myosin VIIA throughout vertebrate evolution and establishes mariner as the first fish model for human hereditary deafness.     

Retention of generalized hair cell patterns in the inner ear of the primitive flatfish psettodes

Flatfish are a group of uniquely asymmetrical vertebrates, lying always on one side. This postural control depends on the vestibular receptors of the inner ear. From the most primitive living flatfish, orientations of sensory hair cells in the inner ear were mapped by scanning electron microscopy. The maps of the three otolith organs, the three semicircular cristae, and the macula neglecta (newly discovered here for flatfish) show patterns that are very similar to those in many upright teleosts, particularly perches. Thus, peripheral sensory structure does not require modification for the unusual postural control of flatfish.     

Interspecific Variations of Inner Ear Structure in the Deep‐Sea Fish Family Melamphaidae

Inner ear structures are compared among three major genera of the deep‐sea fish family Melamphaidae (bigscales and ridgeheads). Substantial interspecific variation is found in the saccular otoliths, including the presence of a unique otolithic “spur” in the genera Melamphaes and Poromitra. The variation in the saccular otolith is correlated with an increase in the number of hair bundle orientation groups on the sensory epithelia from the genera Scopelogadus to Poromitra to Melamphaes. The diverse structural variations found in the saccule may reflect the evolutionary history of these species. The sensory hair cell bundles in this family have the most variable shapes yet encountered in fish ears. In the saccule, most of the hair bundles are 15–20 μm high, an exceptional height for fish otolithic end organs. These bundles have large numbers of stereovilli, including some that reach the length of the kinocilium. In the utricle, the striolar region separates into two unusually shaped areas that have not been described in any other vertebrates. The brains in all species have a relatively small olfactory bulb and optic tectum, as well as an enlarged posterior cerebellar region that is likely to be involved in inner ear and lateral line (octavolateral) functions. Data from melamphaids support the hypothesis that specialized anatomical structures are found in the ears of some (if not most) deep‐sea fishes, presumably enhancing their hearing sensitivity. Anat Rec, 296:1064–1082, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs

The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors. Developmental Dynamics 239:1849–1857, 2010. © 2010 Wiley‐Liss, Inc.     

蝾螈椭圆囊毛细胞换能、编码和突触传递的形态学基础

本实验研究椭圆囊毛细胞换能、编码和突触传递的形态和显微力学基础。以幼年蝾螈为对象，光镜和电镜观察椭圆囊囊斑的微细和超微结构。实验结果：（１）只有毛细胞的高静纤毛和动纤毛的头部和耳石膜接触，耳石膜的剪切力由直接和间接两种途径传递至静纤毛；（２）耳石膜和表皮板组成纤毛束上下两端的致密板状结构，皮板下微管起固定和支撑下板作用，在两板之间的无定形物质有缓冲和利于上板滑动的功能，这种安排是纤毛受力后偏曲的基础；（３）囊斑上皮存在着动纤毛排列方向不同的四种毛细胞，感受器依靠这些分布不同的毛细胞群进行信号编码；（４）多根传入神经末梢和１～２ 根传出神经末梢与毛细胞构成传入和传出突触，存在于毛细胞底部胞液内和传出神经末梢内的囊泡是突触传递的物质基础。     

Afferent innervation patterns of the saccule in pigeons

The innervation patterns of vestibular saccular afferents were quantitatively investigated in pigeons using biotinylated dextran amine as a neural tracer and three-dimensional computer reconstruction. Type I hair cells were found throughout a large portion of the macula, with the highest density observed in the striola. Type II hair cells were located throughout the macula, with the highest density in the extrastriola. Three classes of afferent innervation patterns were observed, including calyx, dimorph, and bouton units, with 137 afferents being anatomically reconstructed and used for quantitative comparisons. Calyx afferents were located primarily in the striola, innervated a number of type I hair cells, and had small innervation areas. Most calyx afferent terminal fields were oriented parallel to the anterior-posterior axis and the morphological polarization reversal line. Dimorph afferents were located throughout the macula, contained fewer type I hair cells in a calyceal terminal than calyx afferents and had medium sized innervation areas. Bouton afferents were restricted to the extrastriola, with multi-branching fibers and large innervation areas. Most of the dimorph and bouton afferents had innervation fields that were oriented dorso-ventrally but were parallel to the neighboring reversal line. The organizational morphology of the saccule was found to be distinctly different from that of the avian utricle or lagena otolith organs and appears to represent a receptor organ undergoing evolutionary adaptation toward sensing linear motion in terrestrial and aerial species.     

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.