A Xenopus neuromast bioassay for chemical ototoxicity

Background: Ototoxic chemicals can impair the senses of hearing and balance in mammals through irreversible damage to the mechanosensory bundles of inner ear hair cells. Fish and amphibians are useful models for investigating ototoxicity because their inner ear hair cells, like those of mammals, are susceptible to damage by ototoxins. Moreover, amphibian mechanosensation is augmented by a lateral line organ on the body surface that comprises external mechanosensory hair cells. The lateral line hair cells are arranged in clusters (neuromasts) and are structurally and functionally similar to inner ear hair cells, but are more accessible for experimental manipulation. Herein, we implemented neuromasts of the amphibian (Xenopus) lateral line as an organ system for evaluating the effects of ototoxic chemicals, such as antibiotics, on mechanosensory hair cell bundles. Methods: We examined the ultrastructure of larval Xenopus laevis neuromasts with scanning electron microscopy (SEM) after larvae were continuously exposed to ototoxic aminoglycoside antibiotics at sub-lethal concentrations (gentamicin; streptomycin; neomycin) for 72 hours. Results: SEM images demonstrated that 72 hours of exposure to antibiotic concentrations greater than 25 μM reduced the hair cell bundle number in lateral line neuromasts. Conclusion: Therapeutic drug studies will benefit from the incorporation of bioassay strategies that evaluate ototoxicity across multiple species including genera of amphibian origin such as Xenopus. Our outcomes support the use of the Xenopus lateral line for identification of potential ototoxic chemicals and suggest that Xenopus neuromast hair cell bundles can withstand antibiotic exposure. The Xenopus bioassay presented here can be incorporated into drug discovery methodology as a high-resolution phenotypic screen for ototoxic effects.

The Hair Cell α9α10 Nicotinic Acetylcholine Receptor: Odd Cousin in an Old Family

Nicotinic acetylcholine receptors (nAChRs) are a subfamily of pentameric ligand-gated ion channels with members identified in most eumetazoan clades. In vertebrates, they are divided into three subgroups, according to their main tissue of expression: neuronal, muscle and hair cell nAChRs. Each receptor subtype is composed of different subunits, encoded by paralogous genes. The latest to be identified are the α9 and α10 subunits, expressed in the mechanosensory hair cells of the inner ear and the lateral line, where they mediate efferent modulation. α9α10 nAChRs are the most divergent amongst all nicotinic receptors, showing marked differences in their degree of sequence conservation, their expression pattern, their subunit co-assembly rules and, most importantly, their functional properties. Here, we review recent advances in the understanding of the structure and evolution of nAChRs. We discuss the functional consequences of sequence divergence and conservation, with special emphasis on the hair cell α9α10 receptor, a seemingly distant cousin of neuronal and muscle nicotinic receptors. Finally, we highlight potential links between the evolution of the octavolateral system and the extreme divergence of vertebrate α9α10 receptors.

Spatial and temporal expression of PORCN is highly dynamic in the developing mouse cochlea.

The mammalian organ of Corti is a highly specialized sensory organ of the cochlea with fine-grained pattern that is essential for auditory function. Previous studies show that the Wnt pathway regulates proliferation, promotes medial compartment formation in the cochlea, differentiation of the mechanosensory hair cells and axon guidance of Type II afferent neurons. WNT ligand expressions are highly dynamic throughout development but are insufficient to explain the roles of the Wnt pathway. We address a potential way for how WNTs specify the medial compartment by characterizing the expression of Porcupine (PORCN), an O-acyltransferase that palmitoylates WNT ligands for secretion. We show PORCN expression across embryonic ages (E)12.5 - E14.5, E16.5, and postnatal day (P)1. Our results showed enriched PORCN in the medial domains during early stages of development, indicating that WNTs have a stronger influence on patterning of the medial compartment. PORCN was rapidly downregulated after E14.5, following the onset of sensory cell differentiation; residual expression remained in some hair cells and supporting cells. On E14.5 and E16.5, we also examined the spatial expression of Gsk3β, an inhibitor of canonical Wnt signaling to determine its potential role in radial patterning of the cochlea. Gsk3β was broadly expressed across the radial axis of the epithelium; therefore, unlikely to control WNT-mediated medial specification. In conclusion, the spatial expression of PORCN enriches WNT secretion from the medial domains of the cochlea to influence the specification of cell fates in the medial sensory domain.

Mechanosensory neuron regeneration in adult Drosophila

Auditory and vestibular mechanosensory hair cells do not regenerate following injury or aging in the adult mammalian inner ear, inducing irreversible hearing loss and balance disorders for millions of people. Research on model systems showing replacement of mechanosensory cells can provide mechanistic insights into developing new regenerative therapies. Here, we developed lineage tracing systems to reveal the generation of mechanosensory neurons in the Johnston's Organ (JO) of intact adult Drosophila, which are the functional counterparts to hair cells in vertebrates. New JO neurons develop cilia and target central brain circuitry. Unexpectedly, mitotic recombination clones point to JO neuron self-replication as a likely source of neuronal plasticity. This mechanism is further enhanced upon treatment with experimental and ototoxic compounds. Our findings introduce a new platform to expedite research on mechanisms and compounds mediating mechanosensory cell regeneration, with nascent implications for hearing and balance restoration.

Supervised machine learning for automated classification of human Wharton’s Jelly cells and mechanosensory hair cells

Tissue engineering and gene therapy strategies offer new ways to repair permanent damage to mechanosensory hair cells (MHCs) by differentiating human Wharton’s Jelly cells (HWJCs). Conventionally, these strategies require the classification of each cell as differentiated or undifferentiated. Automated classification tools, however, may serve as a novel method to rapidly classify these cells. In this paper, images from previous work, where HWJCs were differentiated into MHC-like cells, were examined. Various cell features were extracted from these images, and those which were pertinent to classification were identified. Different machine learning models were then developed, some using all extracted data and some using only certain features. To evaluate model performance, the area under the curve (AUC) of the receiver operating characteristic curve was primarily used. This paper found that limiting algorithms to certain features consistently improved performance. The top performing model, a voting classifier model consisting of two logistic regressions, a support vector machine, and a random forest classifier, obtained an AUC of 0.9638. Ultimately, this paper illustrates the viability of a novel machine learning pipeline to automate the classification of undifferentiated and differentiated cells. In the future, this research could aid in automated strategies that determine the viability of MHC-like cells after differentiation.

Axodendritic versus axosomatic cochlear efferent termination is determined by afferent type in a hierarchical logic of circuit formation

Hearing involves a stereotyped neural network communicating cochlea and brain. How this sensorineural circuit assembles is largely unknown. The cochlea houses two types of mechanosensory hair cells differing in function (sound transmission versus amplification) and location (inner versus outer compartments). Inner (IHCs) and outer hair cells (OHCs) are each innervated by a distinct pair of afferent and efferent neurons: IHCs are contacted by type I afferents receiving axodendritic efferent contacts; OHCs are contacted by type II afferents and axosomatically terminating efferents. Using an Insm1 mouse mutant with IHCs in the position of OHCs, we discover a hierarchical sequence of instructions in which first IHCs attract, and OHCs repel, type I afferents; second, type II afferents innervate hair cells not contacted by type I afferents; and last, afferent fiber type determines if and how efferents innervate, whether axodendritically on the afferent, axosomatically on the hair cell, or not at all.

Inhibition of a transcriptional repressor rescues hearing in a splicing factor–deficient mouse

In mechanosensory hair cells (HCs) of the ear, the transcriptional repressor REST is continuously inactivated by alternative splicing of its pre-mRNA. This mechanism of REST inactivation is crucial for hearing in humans and mice. Rest is one of many pre-mRNAs whose alternative splicing is regulated by the splicing factor SRRM4; Srrm4 loss-of-function mutation in mice (Srrm4bv/bv) causes deafness, balance defects, and degeneration of all HC types other than the outer HCs (OHCs). The specific splicing alterations that drive HC degeneration in Srrm4bv/bv mice are unknown, and the mechanism underlying SRRM4-independent survival of OHCs is undefined. Here, we show that transgenic expression of a dominant-negative REST fragment in Srrm4bv/bv mice is sufficient for long-term rescue of hearing, balancing, HCs, alternative splicing of Rest, and expression of REST target genes including the Srrm4 paralog Srrm3. We also show that in HCs, SRRM3 regulates many of the same exons as SRRM4; OHCs are unique among HCs in that they transiently down-regulate Rest transcription as they mature to express Srrm3 independently of SRRM4; and simultaneous SRRM4–SRRM3 deficiency causes complete HC loss by preventing inactivation of REST in all HCs. Thus, our data reveal that REST inactivation is the primary and essential role of SRRM4 in the ear, and that OHCs differ from other HCs in the SRRM4-independent expression of the functionally SRRM4-like splicing factor SRRM3.

Mechanosensory neuron regeneration in adult Drosophila

ABSTRACTAuditory and vestibular mechanosensory hair cells do not regenerate following injury or aging in the adult mammalian inner ear, inducing irreversible hearing loss and balance disorders for millions of people. Research on model systems showing replacement of mechanosensory cells can provide mechanistic insights into developing new regenerative therapies. Here, we developed lineage tracing systems to reveal, for the first time, the generation of mechanosensory neurons in the Johnston’s Organ (JO) of intact adult Drosophila, which are the functional counterparts to hair cells in vertebrates. New JO neurons develop cilia, express an essential mechano-transducer gene and target central brain circuitry. Furthermore, we identified self-replication of JO neurons as an unexpected mechanism of neuronal plasticity, which is enhanced upon treatment with experimental and ototoxic compounds. Our findings introduce a new platform to expedite research about mechanisms and compounds mediating mechanosensory cell regeneration, with implications for hearing and balance restoration in humans.SUMMARY STATEMENTUsing refined lineage tracing and live imaging, we identified self-renewal of mechanosensory neurons in adult Drosophila, the functional counterparts to vertebrate hair cells, and their enhanced regeneration through pharmacological administration.