Leila Abbas

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

A zebrafish model for Waardenburg syndrome type IV reveals diverse roles for Sox10 in the otic vesicle.

SUMMARY In humans, mutations in the SOX10 gene are a cause of the auditory-pigmentary disorder Waardenburg syndrome type IV (WS4) and related variants. SOX10 encodes an Sry-related HMG box protein essential for the development of the neural crest; deafness in WS4 and other Waardenburg syndromes is usually attributed to loss of neural-crest-derived melanocytes in the stria vascularis of the cochlea. However, SOX10 is strongly expressed in the developing otic vesicle and so direct roles for SOX10 in the otic epithelium might also be important. Here, we examine the otic phenotype of zebrafish sox10 mutants, a model for WS4. As a cochlea is not present in the fish ear, the severe otic phenotype in these mutants cannot be attributed to effects on this tissue. In zebrafish sox10 mutants, we see abnormalities in all otic placodal derivatives. Gene expression studies indicate deregulated expression of several otic genes, including fgf8, in sox10 mutants. Using a combination of mutant and morphant data, we show that the three sox genes belonging to group E (sox9a, sox9b and sox10) provide a link between otic induction pathways and subsequent otic patterning: they act redundantly to maintain sox10 expression throughout otic tissue and to restrict fgf8 expression to anterior macula regions. Single-cell labelling experiments indicate a small and transient neural crest contribution to the zebrafish ear during normal development, but this is unlikely to account for the strong defects seen in the sox10 mutant. We discuss the implication that the deafness in WS4 patients with SOX10 mutations might reflect a haploinsufficiency for SOX10 in the otic epithelium, resulting in patterning and functional abnormalities in the inner ear.

Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene

Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression.

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Endolymph is the specialised extracellular fluid present inside the inner ear. In mammals, disruptions to endolymph homeostasis can result in either collapse or distension of the endolymphatic compartment in the cochlea, with concomitant hearing loss. The zebrafish little ears (lte) mutant shows a collapse of the otic vesicle in the larva, apparently owing to a loss of endolymphatic fluid in the ear, together with an over-inflation of the swim bladder. Mutant larvae display signs of abnormal vestibular function by circling and swimming upside down. The two available alleles of lte are homozygous lethal: mutant larvae fail to thrive beyond 6 days post-fertilisation. Patterning of the otic vesicle is apparently normal. However, the expression of several genes thought to play a role in endolymph production is downregulated, including the sodium-potassium-chloride cotransporter gene nkcc1 (slc12a2) and several Na+/K+-ATPase channel subunit genes. We show here that lte mutations correspond to lesions in nkcc1. Each allele has a point mutation that disrupts splicing, leading to frame shifts in the coding region that predict the generation of truncated products. Endolymph collapse in the lte/nkcc1 mutant shows distinct parallels to that seen in mouse Nkcc1 mutants, validating zebrafish as a model for the study of endolymph disorders. The collapse in ear volume can be ameliorated in the to27d allele of lte by injection of a morpholino that blocks splicing at an ectopic site introduced by the mutation. This exemplifies the use of morpholinos as potential therapeutic agents for genetic disease.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

Non‐technical summary  Due to the conservation of developmental pathways and genetic material over the course of evolution, non‐mammalian ‘model organisms’ such as the zebrafish embryo are emerging as valuable tools to explore causes and potential treatments for human diseases. Ion channels are proteins that form pores and help to establish and control electrical gradients by allowing the flow of ions across biological membranes. A diverse range of key physiological mechanisms in every organ in the body depends on the activity of ion channels. In this paper, we show that a potassium‐selective channel that underlies salt reabsorption and potassium excretion in the human kidney is also expressed in zebrafish in cells that are important regulators of salt balance. Disruption of the channels expression in zebrafish leads to effects on the activity of the heart, consistent with a role for this channel in the control of potassium balance in the embryo.

Aminoglycoside ototoxicity and hair cell ablation in the adult gerbil: A simple model to study hair cell loss and regeneration

The Mongolian gerbil, Meriones unguiculatus, has been widely employed as a model for studies of the inner ear. In spite of its established use for auditory research, no robust protocols to induce ototoxic hair cell damage have been developed for this species. In this paper, we demonstrate the development of an aminoglycoside-induced model of hair cell loss, using kanamycin potentiated by the loop diuretic furosemide. Interestingly, we show that the gerbil is relatively insensitive to gentamicin compared to kanamycin, and that bumetanide is ineffective in potentiating the ototoxicity of the drug. We also examine the pathology of the spiral ganglion after chronic, long-term hair cell damage. Remarkably, there is little or no neuronal loss following the ototoxic insult, even at 8 months post-damage. This is similar to the situation often seen in the human, where functioning neurons can persist even decades after hair cell loss, contrasting with the rapid, secondary degeneration found in rats, mice and other small mammals. We propose that the combination of these factors makes the gerbil a good model for ototoxic damage by induced hair cell loss.

The zebrafish inner ear

Publisher Summary This chapter discusses the development of the zebrafish inner ear. The inner ear arises from an ectodermal thickening, the otic placode, which, like other sensory placodes, originates in a common preplacodal region (PPR) surrounding the neural plate at the end of gastrulation. Specification of the PPR, which is marked by the expression of dlx, eya, six, and irx family genes, is known to be dependent on the correct levels of both bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling. Induction of the otic placode from the PPR occurs during early somite stages in zebrafish. The otic placode is morphologically evident from 16 hours post fertilization, when it begins to cavitate to form an elongated, hollow epithelial ball—that is, the “otic vesicle.” Two of the very earliest patterning events in the otic placode and vesicle are the establishment of the prosensory and proneural domains that give rise to sensory hair cells and neuroblasts, respectively. In the zebrafish otic vesicle, the atoh genes have an early proneural function in establishing the prosensory domain, and are also required later for hair cell differentiation.

09-P020 Nkcc1/Slc12a2 is required for the regulation of endolymph in the otic vesicle and volume of the swim bladder in the zebrafish larva

base. The anterior cranial base shows an abnormal differentiation of chondrocytes in the mutant, with reduced proliferation and increased terminal differentiation. The main factors responsible to restrict Six2 function to the anterior portion of the cranial base are the tissue-specific transcription of the gene and the presence of compensatory effects from other Six family members, in particular Six1 and Six4. Gain of function experiments indicate that Six2 has a general effect on endochondral bone formation and can affect cartilage development and growth in other body areas. It is likely that Six2 acts by controlling general regulators of chondrocyte differentiation. Our data suggest that Six2 acts to control locally the level of the IGF-axis.

03-P076 Development of semicircular canals in the zebrafish inner ear

We are using zebrafish as a model system for human deafness and vestibular disorders. big ears (bge) is an adult viable zebrafish mutation isolated in a large-scale mutagenesis screen. The lauscher (lau) mutant is allelic to bge by complementation assay. The inner ears of these mutants become swollen at the end of the third day of embryogenesis. Epithelial projections forming the semicircular canal system show morphological and gene expression abnormalities and fail to fuse correctly. Preliminary data suggest that the swelling is a secondary result of the failure of the projections to fuse in the mutant. We have identified the bge gene by genetic mapping and a candidate gene approach. Orthologous genes have been cloned in the human and mouse, but their function is still unknown. Expression of bge appears to be ear-specific, and is restricted to the epithelial projections of the developing semicircular canal system. Point mutations were found in cDNA and genomic DNA for bge and lau, and have been confirmed by PCR-based genotyping assays. Both are missense mutations that predict the substitution of conserved amino acid residues in the protein. A splice site morpholino designed to knock down function of the bge gene phenocopies the semicircular canal projection defects seen in the mutant.

09-P072 Development of the endolymphatic duct and regulation of endolymph production in the zebrafish otic vesicle

The liver forms as part of digestive system. Despite the essential functions of the liver, little is known about its early embryonic development. The clamped s819 mutant was identified in a forward genetic screen for mutations causing defects in endodermal organogenesis in zebrafish. At 44 h post-fertilisation (hpf), clamped s819 mutant embryos display liver hypoplasia and defects in extrahepatic duct morphology. Examination of gene expression indicative of liver specification and differentiation in mutant embryos revealed that specification occurs normally while subsequent differentiation and growth are altered. Similar defects were observed in the developing eyes and fins. Interestingly, in clamped s819 mutants cell death was detected from 40 hpf onwards in the liver abutting mesoderm, and from 48 hpf onwards within the liver and extrahepatic duct. Concomitantly, cell proliferation in the liver of clamped s819 mutants was severely impaired from 44 hpf onwards. Therefore, we hypothesise that Clamped plays a role in promoting hepatic differentiation. Furthermore, Clamped may be required for cross-talk between the mesoderm and endoderm at this stage of liver organogenesis. Mosaic analysis will be carried out to test the tissue autonomous function of Clamped within this process. Positional cloning has mapped the molecular lesion underlying the clamped s819 mutant phenotype to a 240 kb interval on linkage group 14, containing three candidate genes. None of the candidates have so far been implicated in liver organogenesis, thus investigating their function promises to further our understanding of liver differentiation and growth.