ISL1 is necessary for auditory neuron development and contributes toward tonotopic organization.

A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.

Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification.

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Early Deletion of Neurod1 Alters Neuronal Lineage Potential and Diminishes Neurogenesis in the Inner Ear.

Neuronal development in the inner ear is initiated by expression of the proneural basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin1 that specifies neuronal precursors in the otocyst. The initial specification of the neuroblasts within the otic epithelium is followed by the expression of an additional bHLH factor, Neurod1. Although NEUROD1 is essential for inner ear neuronal development, the different aspects of the temporal and spatial requirements of NEUROD1 for the inner ear and, mainly, for auditory neuron development are not fully understood. In this study, using Foxg1Cre for the early elimination of Neurod1 in the mouse otocyst, we showed that Neurod1 deletion results in a massive reduction of differentiating neurons in the otic ganglion at E10.5, and in the diminished vestibular and rudimental spiral ganglia at E13.5. Attenuated neuronal development was associated with reduced and disorganized sensory epithelia, formation of ectopic hair cells, and the shortened cochlea in the inner ear. Central projections of inner ear neurons with conditional Neurod1 deletion are reduced, unsegregated, disorganized, and interconnecting the vestibular and auditory systems. In line with decreased afferent input from auditory neurons, the volume of cochlear nuclei was reduced by 60% in Neurod1 mutant mice. Finally, our data demonstrate that early elimination of Neurod1 affects the neuronal lineage potential and alters the generation of inner ear neurons and cochlear afferents with a profound effect on the first auditory nuclei, the cochlear nuclei.

Overexpression of Isl1 under the Pax2 Promoter, Leads to Impaired Sound Processing and Increased Inhibition in the Inferior Colliculus.

The LIM homeodomain transcription factor ISL1 is essential for the different aspects of neuronal development and maintenance. In order to study the role of ISL1 in the auditory system, we generated a transgenic mouse (Tg) expressing Isl1 under the Pax2 promoter control. We previously reported a progressive age-related decline in hearing and abnormalities in the inner ear, medial olivocochlear system, and auditory midbrain of these Tg mice. In this study, we investigated how Isl1 overexpression affects sound processing by the neurons of the inferior colliculus (IC). We recorded extracellular neuronal activity and analyzed the responses of IC neurons to broadband noise, clicks, pure tones, two-tone stimulation and frequency-modulated sounds. We found that Tg animals showed a higher inhibition as displayed by two-tone stimulation; they exhibited a wider dynamic range, lower spontaneous firing rate, longer first spike latency and, in the processing of frequency modulated sounds, showed a prevalence of high-frequency inhibition. Functional changes were accompanied by a decreased number of calretinin and parvalbumin positive neurons, and an increased expression of vesicular GABA/glycine transporter and calbindin in the IC of Tg mice, compared to wild type animals. The results further characterize abnormal sound processing in the IC of Tg mice and demonstrate that major changes occur on the side of inhibition.

Combined Atoh1 and Neurod1 Deletion Reveals Autonomous Growth of Auditory Nerve Fibers.

Ear development requires the transcription factors ATOH1 for hair cell differentiation and NEUROD1 for sensory neuron development. In addition, NEUROD1 negatively regulates Atoh1 gene expression. As we previously showed that deletion of the Neurod1 gene in the cochlea results in axon guidance defects and excessive peripheral innervation of the sensory epithelium, we hypothesized that some of the innervation defects may be a result of abnormalities in NEUROD1 and ATOH1 interactions. To characterize the interdependency of ATOH1 and NEUROD1 in inner ear development, we generated a new Atoh1/Neurod1 double null conditional deletion mutant. Through careful comparison of the effects of single Atoh1 or Neurod1 gene deletion with combined double Atoh1 and Neurod1 deletion, we demonstrate that NEUROD1-ATOH1 interactions are not important for the Neurod1 null innervation phenotype. We report that neurons lacking Neurod1 can innervate the flat epithelium without any sensory hair cells or supporting cells left after Atoh1 deletion, indicating that neurons with Neurod1 deletion do not require the presence of hair cells for axon growth. Moreover, transcriptome analysis identified genes encoding axon guidance and neurite growth molecules that are dysregulated in the Neurod1 deletion mutant. Taken together, we demonstrate that much of the projections of NEUROD1-deprived inner ear sensory neurons are regulated cell-autonomously.