Identification of cellular, functional and genetic causes of Hoxb1null sensorineural hearing loss

The assembly of central auditory subcircuits depends on the spatially and temporally ordered sequence of specification, migration and connectivity. Alterations of one of these events will lead to abnormal circuit formation and different types of deafness. Recessive mutations in the human homeobox HOXB1 gene cause sensorineural hearing impairments characterized by increased auditory threshold and loss of distortion product otoacoustic emissions, mainly due to abnormal cochlear activity of outer hair cells (OHCs) and absence of sound amplification Our studies in mouse show that Hoxb1 is essential for the specification of rhombomere 4 (r4)-derived auditory sensory and motor neurons contributing to the sound transmission pathway and to the establishment of sensorimotor reflex circuits. Hoxb1null mice have abnormal r4-derived sensory nuclei, lack inner ear efferent motor neurons and show degeneration of OHCs. This leads to defective cochlear amplification and altered auditory thresholds that well replicate the hearing loss observed in patients with mutations in the HOXB1 gene. However, the exact mechanisms leading to the auditory impairments described in mice and patients are not known. Hoxb1 is expressed in the medial olivocochlear (MOC) neurons that innervate the OHCs, but not in cochlear hair cells. In Hoxb1null mice, MOC neurons fail to be generated, but morphology of OHCs is not affected until P8, when MOC/OHC interactions normally occur; however, OHCs start to degenerate later when these interactions fail to be established, with consequent appearance of threshold impairments. Interesting, the persistence of few MOC neurons in Hoxb1flox b1r4-Cre mutants, in which Hoxb1 is inactivated only in r4, correlates well with less severe OHC morphology and threshold defects. To directly assess whether the degeneration of OHCs and the increased hearing threshold observed in Hoxb1null mutants depends on the absence of MOC innervation, or alternatively is due to affected sensory r4-derived components, we independently altered Hoxb1 function in r4 motor or sensory neurons. The OC efferent bundles were genetically deleted by using Hoxb1flox Nkx2.2-Cre mutants, in which Hoxb1 is exclusively inactivated in r4 motor neurons, whereas the sensory glutamatergic and GABAergic/glycinergic cochlear populations were affected in Hoxb1flox Atoh1-Cre and Hoxb1flox Ptf1a-Cre mice. By combining anatomical, electrophysiological and molecular analyses, we demonstrated a key role for MOC neurons on OHC survival and sound amplification as the major cause for the Hoxb1 phenotype. Overall, these data show that during a critical postnatal period interactions between MOC neurons and OHCs are crucial for establishing the correct sound amplification.