Comparative expression analysis of the Atoh7 gene regulatory network in the mouse and chicken auditory hindbrain.

The mammalian and avian auditory brainstem likely arose by independent evolution. To compare the underlying molecular mechanisms, we focused on Atoh7, as its expression pattern in the mammalian hindbrain is restricted to bushy cells in the ventral cochlear nucleus. We thereby took advantage of an Atoh7 centered gene regulatory network (GRN) in the retina including upstream regulators, Hes1 and Pax6, and downstream targets, Ebf3 and Eya2. In situ hybridization demonstrated for the latter four genes broad expression in all three murine cochlear nuclei at postnatal days (P) 4 and P30, contrasting the restricted expression of Atoh7. In chicken, all five transcription factors were expressed in all auditory hindbrain nuclei at embryonic day (E) 13 and P14. Notably, all five genes showed graded expression in the embryonic nucleus magnocellularis (NM). Atoh7 was highly expressed in caudally located neurons, whereas the other four transcription factors were highly expressed in rostrally located neurons. Thus, Atoh7 shows a strikingly different expression between the mammalian and avian auditory hindbrain. This together with the consistent absence of graded expression of GRN components in developing mammalian nuclei provide the first molecular support to the current view of convergent evolution as a major mechanism in the amniote auditory hindbrain. The graded expression of five transcription factors specifically in the developing NM confirms this nucleus as a central organizer of tonotopic features in birds. Finally, the expression of all five retinal GRN components in the auditory system suggests co-options of genes for development of sensory systems of distinct modalities.

Shared and organ-specific gene-expression programs during the development of the cochlea and the superior olivary complex.

The peripheral and central auditory subsystems together form a complex sensory network that allows an organism to hear. The genetic programs of the two subsystems must therefore be tightly coordinated during development. Yet, their interactions and common expression pathways have never been systematically explored. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and are essential for normal development of the auditory system. We performed mRNA and small-RNA sequencing of organs from both auditory subsystems at three critical developmental timepoints (E16, P0, P16) to obtain a comprehensive and unbiased insight of their expression profiles. Our analysis reveals common and organ-specific expression patterns for differentially regulated mRNAs and miRNAs, which could be clustered with a particular selection of functions such as inner ear development, Wnt signalling, K+ transport, and axon guidance, based on gene ontology. Bioinformatics detected enrichment of predicted targets of specific miRNAs in the clusters and predicted regulatory interactions by monitoring opposite trends of expression of miRNAs and their targets. This approach identified six miRNAs as strong regulatory candidates for both subsystems. Among them was miR-96, an established critical factor for proper development in both subsystems, demonstrating the strength of our approach. We suggest that other miRNAs identified by this analysis are also common effectors of proper hearing acquirement. This first combined comprehensive analysis of the developmental program of the peripheral and central auditory systems provides important data and bioinformatics insights into the shared genetic program of the two sensory subsystems and their regulation by miRNAs.

Loss of miR-183/96 Alters Synaptic Strength via Presynaptic and Postsynaptic Mechanisms at a Central Synapse.

A point mutation in miR-96 causes non-syndromic progressive peripheral hearing loss and alters structure and physiology of the central auditory system. To gain further insight into the functions of microRNAs (miRNAs) within the central auditory system, we investigated constitutive Mir-183/96dko mice of both sexes. In this mouse model, the genomically clustered miR-183 and miR-96 are constitutively deleted. It shows significantly and specifically reduced volumes of auditory hindbrain nuclei, because of decreases in cell number and soma size. Electrophysiological analysis of the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) demonstrated strongly altered synaptic transmission in young-adult mice. We observed an increase in quantal content and readily releasable vesicle pool size in the presynapse while the overall morphology of the calyx was unchanged. Detailed analysis of the active zones (AZs) revealed differences in its molecular composition and synaptic vesicle (SV) distribution. Postsynaptically, altered clustering and increased synaptic abundancy of the AMPA receptor subunit GluA1 was observed resulting in an increase in quantal amplitude. Together, these presynaptic and postsynaptic alterations led to a 2-fold increase of the evoked excitatory postsynaptic currents in MNTB neurons. None of these changes were observed in deaf Cldn14ko mice, confirming an on-site role of miR-183 and miR-96 in the auditory hindbrain. Our data suggest that the Mir-183/96 cluster plays a key role for proper synaptic transmission at the calyx of Held and for the development of the auditory hindbrain.SIGNIFICANCE STATEMENT The calyx of Held is the outstanding model system to study basic synaptic physiology. Yet, genetic factors driving its morphologic and functional maturation are largely unknown. Here, we identify the Mir-183/96 cluster as an important factor to regulate its synaptic strength. Presynaptically, Mir-183/96dko calyces show an increase in release-ready synaptic vesicles (SVs), quantal content and abundance of the proteins Bassoon and Piccolo. Postsynaptically, the quantal size as well as number and size of GluA1 puncta were increased. The two microRNAs (miRNAs) are thus attractive candidates for regulation of synaptic maturation and long-term adaptations to sound levels. Moreover, the different phenotypic outcomes of different types of mutations in the Mir-183 cluster corroborate the requirement of mutation-tailored therapies in patients with hearing loss.

Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transport.

K+-Cl- cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell-volume regulation. KCCs exhibit significant variations in K+ affinities, yet recent atomic structures demonstrated that K+- and Cl--binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Here, we used the extracellular loop 2 (EL2) that links transmembrane helices (TMs) 3 and 4, as a mechanism to modulate ECD folding. We compared consequences of point mutations in the K+-binding site on the function of WT KCC2 and in a KCC2 variant, in which EL2 was structurally altered by insertion of a IFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHAAA (3xHA) tag (36 amino acids). In WT KCC2, individual mutations of five residues in the K+-binding site resulted in a 2- to 3-fold decreased transport rate. However, the same mutations in the KCC2 variant with EL2 structurally altered by insertion of a 3xHA tag had no effect on transport activity. Homology models of mouse KCC2 with the 3xHA tag inserted into EL2 using ab initio prediction were generated. The models suggest subtle conformational changes occur in the ECD upon EL2 modification. These data suggest that a conformational change in the ECD, for example, by interaction with EL2, might be an elegant way to modulate the K+ affinity of the different isoforms in the KCC subfamily.

Expression pattern of cochlear microRNAs in the mammalian auditory hindbrain.

The auditory system comprises the auditory periphery, engaged in sound transduction and the central auditory system, implicated in auditory information processing and perception. Recently, evidence mounted that the mammalian peripheral and central auditory systems share a number of genes critical for proper development and function. This bears implication for auditory rehabilitation and evolution of the auditory system. To analyze to which extent microRNAs (miRNAs) belong to genes shared between both systems, we characterize the expression pattern of 12 cochlea-abundant miRNAs in the central auditory system. Quantitative real-time PCR (qRT-PCR) demonstrated expression of all 12 genes in the cochlea, the auditory hindbrain and the non-auditory prefrontal cortex (PFC) at embryonic stage (E)16 and postnatal stages (P)0 and P30. Eleven of them showed differences in expression between tissues and nine between the developmental time points. Hierarchical cluster analysis revealed that the temporal expression pattern in the auditory hindbrain was more similar to the PFC than to the cochlea. Spatiotemporal expression analysis by RNA in situ hybridization demonstrated widespread expression throughout the cochlear nucleus complex (CNC) and the superior olivary complex (SOC) during postnatal development. Altogether, our data indicate that miRNAs represent a relevant class of genetic factors functioning across the auditory system. Given the importance of gene regulatory network (GRN) components for development, physiology and evolution, the 12 miRNAs provide promising entry points to gain insights into their molecular underpinnings in the auditory system.

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.

Phosphoregulation of the intracellular termini of K+-Cl- cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Evolution of Endolymph Secretion and Endolymphatic Potential Generation in the Vertebrate Inner Ear.

The ear of extant vertebrates reflects multiple independent evolutionary trajectories. Examples include the middle ear or the unique specializations of the mammalian cochlea. Another striking difference between vertebrate inner ears concerns the differences in the magnitude of the endolymphatic potential. This differs both between the vestibular and auditory part of the inner ear as well as between the auditory periphery in different vertebrates. Here we provide a comparison of the cellular and molecular mechanisms in different endorgans across vertebrates. We begin with the lateral line and vestibular systems, as they likely represent plesiomorphic conditions, then review the situation in different vertebrate auditory endorgans. All three systems harbor hair cells bathed in a high (K+) environment. Superficial lateral line neuromasts are bathed in an electrogenically maintained high (K+) microenvironment provided by the complex gelatinous cupula. This is associated with a positive endocupular potential. Whether this is a special or a universal feature of lateral line and possibly vestibular cupulae remains to be discovered. The vestibular system represents a closed system with an endolymph that is characterized by an enhanced (K+) relative to the perilymph. Yet only in land vertebrates does (K+) exceed (Na+). The endolymphatic potential ranges from +1 to +11 mV, albeit we note intriguing reports of substantially higher potentials of up to +70 mV in the cupula of ampullae of the semicircular canals. Similarly, in the auditory system, a high (K+) is observed. However, in contrast to the vestibular system, the positive endolymphatic potential varies more substantially between vertebrates, ranging from near zero mV to approximately +100 mV. The tissues generating endolymph in the inner ear show considerable differences in cell types and location. So-called dark cells and the possibly homologous ionocytes in fish appear to be the common elements, but there is always at least one additional cell type present. To inspire research in this field, we propose a classification for these cell types and discuss potential evolutionary relationships. Their molecular repertoire is largely unknown and provides further fertile ground for future investigation. Finally, we propose that the ultimate selective pressure for an increased endolymphatic potential, as observed in mammals and to a lesser extent in birds, is specifically to maintain the AC component of the hair-cell receptor potential at high frequencies. In summary, we identify intriguing questions for future directions of research into the molecular and cellular basis of the endolymph in the different compartments of the inner ear. The answers will provide important insights into evolutionary and developmental processes in a sensory organ essential to many species, including humans.

Activity-dependent formation of a vesicular inhibitory amino acid transporter gradient in the superior olivary complex of NMRI mice.

BACKGROUND: In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem. RESULTS: Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient. CONCLUSIONS: Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular factors involved in activity-dependent developmental processes in the auditory system. This would provide an important step forward concerning improved auditory rehabilitation in cases of congenital deafness.

Differences in molecular mechanisms of K+ clearance in the auditory sensory epithelium of birds and mammals.

Mechanoelectrical transduction in the vertebrate inner ear is a highly conserved mechanism that is dependent on K+ influx into hair cells. Here, we investigated the molecular underpinnings of subsequent K+ recycling in the chicken basilar papilla and compared them with those in the mammalian auditory sensory epithelium. As in mammals, the avian auditory hair cell uses KCNQ4, KCNMA1 and KCNMB1 in its K+ efflux system. Expression of KCNQ1 and KCNE1 suggests an additional efflux apparatus in avian hair cells. Marked differences were observed for K+ clearance. In mammals, KCC3, KCC4, Kir4.1 and CLC-K are present in supporting cells. Of these, only CLC-K is expressed in avian supporting cells. Instead, they possess NKCC1 to move K+ across the membrane. This expression pattern suggests an avian clearance mechanism reminiscent of the well-established K+ uptake apparatus present in inner ear secretory cells. Altogether, tetrapod hair cells show similar mechanisms and supporting cells show distinct molecular underpinnings of K+ recycling.

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

Differential patterns of histone methylase EHMT2 and its catalyzed histone modifications H3K9me1 and H3K9me2 during maturation of central auditory system.

Histone methylation is an important epigenetic mark leading to changes in DNA accessibility and transcription. Here, we investigate immunoreactivity against the euchromatic histone-lysine N-methyltransferase EHMT2 and its catalyzed mono- and dimethylation marks at histone 3 lysine 9 (H3K9me1 and H3K9me2) during postnatal differentiation of the mouse central auditory system. In the brainstem, expression of EHMT2 was high in the first postnatal week and down-regulated thereafter. In contrast, immunoreactivity in the auditory cortex (AC) remained high during the first year of life. This difference might be related to distinct demands for adult plasticity. Analyses of two deaf mouse models, namely Cldn14 (-/-) and Cacna1d (-/-), demonstrated that sound-driven or spontaneous activity had no influence on EHMT2 immunoreactivity. The methylation marks H3K9me1 and H3K9me2 were high throughout the auditory system up to 1 year. Young auditory neurons showed immunoreactivity against both methylations at similar intensities, whereas many mature neurons showed stronger labeling for either H3K9me1 or H3K9me2. These differences were only poorly correlated with cell types. To identify methyltransferases contributing to the persistent H3K9me1 and H3K9me2 marks in the adult brainstem, EHMT1 and the retinoblastoma-interacting zinc-finger protein RIZ1 were analyzed. Both were down-regulated during brainstem development, similar to EHMT2. Contrary to EHMT2, EHMT1 was also down-regulated in adult cortical areas. Together, our data reveal a marked difference in EHMT2 levels between mature brainstem and cortical areas and a decoupling between EHMT2 abundance and histone 3 lysine 9 methylations during brainstem differentiation. Furthermore, EHMT1 and EHMT2 are differentially expressed in cortical areas.

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 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.

A novel regulatory locus of phosphorylation in the C terminus of the potassium chloride cotransporter KCC2 that interferes with N-ethylmaleimide or staurosporine-mediated activation.

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser(25), Ser(26), Ser(937), Ser(1022), Ser(1025), and Ser(1026). Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2(WT)). Tl(+) flux measurements demonstrated unchanged transport activity for Ser(25), Ser(26), Ser(1022), Ser(1025), and Ser(1026) mutants. In contrast, KCC2(S937D), mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr(934), a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2(T934D) and KCC2(S937D) mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2(WT), KCC2(T934A), and KCC2(S937A) were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2(T934D) and KCC2(S937D) compared with KCC2(WT). These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.

Evolution of the cation chloride cotransporter family: ancient origins, gene losses, and subfunctionalization through duplication.

The cation chloride cotransporter (CCCs) family comprises of four subfamilies-K(+)-Cl(-) cotransporters (KCCs), Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), and Na(+)-Cl(-) cotransporters (NCCs)-and possibly two additional members-CCC interacting protein (CIP1) and polyamine transporters (CCC9)-as well. Altogether, CCCs can play essential physiological roles in transepithelial ion reabsorption and secretion, cell volume regulation, and inhibitory neurotransmission and so are present across all domains of life. To gain insight into the evolution of this family, we performed a comprehensive phylogenetic analysis using publically available genomic information. Our results clearly support CIP1 as being a true CCC based on shared evolutionary history. By contrast, the status of CCC9 in this regard remains equivocal. We also reveal the existence of a single ancestral CCC gene present in Archaea, from which numerous duplication events at the base of archaeans and eukaryotes lead to the divergence and subsequent neofunctionalization of the paralogous CCC subfamilies. A diversity of ensuing gene-loss events resulted in the complex distribution of CCCs present across the different taxa. Importantly, the occurrence of KCCs in "basal" metazoan taxa like sponges would allow an early formation of fast hyperpolarizing neurotransmission in metazoans. Gene duplications within the CCC subfamilies in vertebrates (in particular, KCCs, NKCCs, and NCCs) lend further evidence to the 2R hypothesis of two rounds of genome duplication at the base of the vertebrate lineage, especially in concert with our syntenic cluster analyses. This increased number of KCCs, NKCCs, and NCCs isoforms facilitates their further, important subfunctionalization in the vertebrate lineage.

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.

Time-dependent gene expression analysis of the developing superior olivary complex.

The superior olivary complex (SOC) is an essential auditory brainstem relay involved in sound localization. To identify the genetic program underlying its maturation, we profiled the rat SOC transcriptome at postnatal days 0, 4, 16, and 25 (P0, P4, P16, and P25, respectively), using genome-wide microarrays (41,012 oligonucleotides (oligos)). Differences in gene expression between two consecutive stages were highest between P4 and P16 (3.6%) and dropped to 0.06% between P16 and P25. To identify SOC-related genetic programs, we also profiled the entire brain at P4 and P25. The number of differentially expressed oligonucleotides between SOC and brain almost doubled from P4 to P25 (4.4% versus 7.6%). These data demonstrate considerable molecular specification around hearing onset, which is rapidly finalized. Prior to hearing onset, several transcription factors associated with the peripheral auditory system were up-regulated, probably coordinating the development of the auditory system. Additionally, crystallin-γ subunits and serotonin-related genes were highly expressed. The molecular repertoire of mature neurons was sculpted by SOC-related up- and down-regulation of voltage-gated channels and G-proteins. Comparison with the brain revealed a significant enrichment of hearing impairment-related oligos in the SOC (26 in the SOC, only 11 in the brain). Furthermore, 29 of 453 SOC-related oligos mapped within 19 genetic intervals associated with hearing impairment. Together, we identified sequential genetic programs in the SOC, thereby pinpointing candidates that may guide its development and ensure proper function. The enrichment of hearing impairment-related genes in the SOC may have implications for restoring hearing because central auditory structures might be more severely affected than previously appreciated.

Retrocochlear function of the peripheral deafness gene Cacna1d.

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.