miR-96 is required for normal development of the auditory hindbrain.

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.

Comparative Analysis of Gene Regulatory Network Components in the Auditory Hindbrain of Mice and Chicken.

The neurons in the mammalian and avian auditory hindbrain nuclei share a number of significant morphological and physiological properties for fast, secure and precise neurotransmission, such as giant synapses, voltage-gated K+ channels and fast AMPA receptors. Based on the independent evolution of the middle ear in these two vertebrate lineages, on different embryonic origins of the nuclei and on marked differences on the circuit level, these similarities are assumed to reflect convergent evolution. Independent acquisition of similar phenotypes can be produced by divergent evolution of genetic mechanisms or by similar molecular mechanisms. The distinction between these two possibilities requires knowledge of the gene regulatory networks (GRNs) that orchestrate the development of auditory hindbrain structures. We therefore compared the expression pattern of GRN components, both transcription factors (TFs) and noncoding RNA, during terminal differentiation of the auditory hindbrain structures in mouse and chicken when neurons acquire their final morphological and electrophysiological properties. In general, we observed broad expression of these genes in the mouse auditory cochlear nucleus complex and the superior olivary complex at both postnatal day 4 (P4) and at P25, and for the chicken at the equivalent developmental stages, i.e. embryonic day 13 (E13) and at P14-P17. Our data are in agreement with a model based on similar molecular mechanisms underlying terminal differentiation and maintenance of neuronal cell identity in the auditory hindbrain of different vertebrate lineages. This conservation might reflect developmental constraints arising from the tagmatic organization of rhombomeres and the evolutionarily highly conserved GRNs operating in these structures.

The emerging framework of mammalian auditory hindbrain development.

A defining feature of the mammalian auditory system is the extensive processing of sound information in numerous ultrafast and temporally precise circuits in the hindbrain. By exploiting the experimental advantages of mouse genetics, recent years have witnessed an impressive advance in our understanding of developmental mechanisms involved in the formation and refinement of these circuits. Here, we will summarize the progress made in four major fields: the dissection of the rhombomeric origins of auditory hindbrain nuclei; the molecular repertoire involved in circuit formation such as Hox transcription factors and the Eph-ephrin signaling system; the timeline of functional circuit assembly; and the critical role of spontaneous activity for circuit refinement. In total, this information provides a solid framework for further exploration of the factors shaping auditory hindbrain circuits and their specializations. A comprehensive understanding of the developmental pathways and instructive factors will also offer important clues to the causes and consequences of hearing-loss related disorders, which represent the most common sensory impairment in humans.

The gene regulatory networks underlying formation of the auditory hindbrain.

Development and evolution of auditory hindbrain nuclei are two major unsolved issues in hearing research. Recent characterization of transgenic mice identified the rhombomeric origins of mammalian auditory nuclei and unraveled genes involved in their formation. Here, we provide an overview on these data by assembling them into rhombomere-specific gene regulatory networks (GRNs), as they underlie developmental and evolutionary processes. To explore evolutionary mechanisms, we compare the GRNs operating in the mammalian auditory hindbrain with data available from the inner ear and other vertebrate groups. Finally, we propose that the availability of genomic sequences from all major vertebrate taxa and novel genetic techniques for non-model organisms provide an unprecedented opportunity to investigate development and evolution of the auditory hindbrain by comparative molecular approaches. The dissection of the molecular mechanisms leading to auditory structures will also provide an important framework for auditory processing disorders, a clinical problem difficult to tackle so far. These data will, therefore, foster basic and clinical hearing research alike.

Evolutionary conservation of Kv3.1 in the barn owl Tyto alba.

For prey capture in the dark, the barn owl Tyto alba has evolved into an auditory specialist with an exquisite capability of sound localization. Adaptations include asymmetrical ears, enlarged auditory processing centers, the utilization of minute interaural time differences, and phase locking along the entire hearing range up to 10 kHz. Adaptations on the molecular level have not yet been investigated. Here, we tested the hypothesis that divergence in the amino acid sequence of the voltage-gated K(+) channel Kv3.1 contributes to the accuracy and high firing rates of auditory neurons in the barn owl. We therefore cloned both splice variants of Kcnc1, the gene encoding Kv3.1. Both splice variants, Kcnc1a and Kcnc1b, encode amino acids identical to those of the chicken, an auditory generalist. Expression analyses confirmed neural-restricted expression of the channel. In summary, our data reveal strong evolutionary conservation of Kcnc1 in the barn owl and point to other genes involved in auditory specializations of this animal. The data also demonstrate the feasibility to address neuroethological questions in organisms with no reference genome by molecular approaches. This will open new avenues for neuroethologists working in these organisms.