Tizzano, Monica (2018) Genetic dissection of Hoxb1 function in the developing mouse auditory and vestibular system rhombomere 4-derived. [Tesi di dottorato]

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Abstract

Rhombomere (r4) and Hox associated genes Hoxb1 and Hoxb2 contribute to the formation of specific auditory and vestibular subcircuits. In particular, sensory and motor components of the sound transmission pathway, sensorimotor reflex circuits, as well as the hindlimb vestibule-spinal reflex, derive from r4 and are strongly affected in Hoxb1 mutants (Di Bonito et al., 2013). Inner ear efferent (IEE) neurons also originate from r4 and form vestibular (VEN) and cochlear (CEN) efferent neuron subpopulations. The CEN is further subdivided into medial (MOC) and lateral (LOC) olivo-cochlear motoneurons. MOC neurons inhibit the motility of outer hair cells (OHCs), which amplify low intensity sounds, while LOCs innervate the afferent terminations on the inner hair cells (IHC) modulating the excitability of the cochlear nerve, thus protecting the cochlea from acoustic damage. Hoxb1null mutants lack MOC and LOC efferent neurons leading to defects in OHCs and in cochlear amplification, and mice have increasing auditory thresholds (Di Bonito et al., 2013). Our hypothesis is that MOC neuron endings play a trophic function on OHCs and that the physical interaction between MOC efferents and OHCs is essential for proper maturation and functioning of OHCs. To exclude a possible contribute of sensory auditory neurons at this phenotype, we performed analysis of conditional Hoxb1 mutants (Ptf1acre Hoxb1 flox/flox, Atoh1cre Hoxb1 flox/flox) where Hoxb1 expression were eliminated in the dorsal region of rhombomere 4 and the sensory structures involved in the acoustic pathway, were selective affected. Our data show that when the ventral domain, where the motoneurons MOC and LOC develop, normally expresses Hoxb1 the OHCs show a regular morphology, even if Hoxb1 expression is affected in the dorsal sensory auditory neurons. So, our data suggest that MOCs could play an important postnatal role but to confirm this hypothesis further investigations are needed using a Cre specific line that selectively eliminate the Hoxb1 expression in the ventral domain of motoneurons. Regarding the vestibular system, Hoxb1null mutant mice also fail to form the VEN at early developmental stages (Di Bonito et al., 2015). However, transmission electron microscopy (TEM) in adult mice reveals the presence of both afferent and efferent neuronal endings on receptor cells. To understand whether projections are missing at birth and new connections gradually appear during the first month by compensatory plasticity mechanisms, we analyzed newborn mutant pups for the presence of efferent endings on hair cells by TEM. Our analysis showed that this mutant fail to develop efferent terminations corroborating our hypothesis that these endings derive from other district and could act as a compensatory mechanism. So, to further confirm this hypothesis we aim to use retrograde labelling in 3-month old mutant mice to assess their eventual presence and identify their origin.

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