Understanding the mechanisms of deafness by using intersectional genetics and functional analysis in the mouse – DEAF

This study involves two research groups with two perfectly complementary expertises, a developmental neurobiology and an auditory neurophysiology and behaviour team. Thanks to this synergy, we are using a series of mouse strains to precisely map and functionally alter the neuronal subtypes originating from distinct subdomains within rhombomere 4 (r4) in the developing hindbrain. These tools will also allow us to use genomic approaches for identifying novel interactors and molecular players involved in setting up r4-specific auditory circuits. We are combining this approach with functional electrophysiological recording from the cochlea and within the cochlea, such as auditory brainstem recording (ABR), electrocochleography and patch-clamp. Overall, we are combining genetic, anatomical and electrophysiological approaches on live animals that will give more insights into how subpopulations and/or subcircuits of the auditory system contribute to the logic of sound encoding and how their alterations determine the different types of deafness. Moreover, our genetic intersectional strategy will help in identifying candidate genes involved in the specification of different functional auditory subcircuits that could be used in future screening for mutations in patients affected by hearing loss. We deeply think that our study can contribute to further understand the genetic, developmental, anatomical causes of different form of hearing loss with the final aim to develop new therapies for patients.

We have now obtained all the crossing of the different mouse strains necessary for the intersectional genetic strategy and functional analysis. We have also validated the activity of the r4-Flippase line by crossing the b1r4Flpo and b1r4Cre lines with the dual reporter and assessing that cells in r4 were properly expressing the GFP. In parallel, we have tested the Cre lines of different dorso-ventral (DV) domains with a Cre-specific reporter mouse, which has helped us in characterizing all their derivatives. Then, the several lines have been crossed to obtain the correct genotype to genetically label distinct auditory DV sub-populations specific to r4. We are following their cell migration and axonal projections at different stages by using specific markers for the distinct type of neurons, as planned in the project.

While waiting for the correct mouse genotype, we have finalized the full description of all r4-derivatives by using the r4-Cre mouse line crossed to the YFP-reporter line. This will not only be very useful for the whole community working on the brainstem and auditory system, but it also represents an anatomical and cellular basis crucial for identifying subpopulations of specific DV r4 subdomains. This study has now been published in the peer-reviewed Journal Brain Structure and Function.

Our partner at the INM is currently investigating the role of the cholinergic olivocochlear bundle in the cochlea using the Hoxb1null mouse model. They confirm that adult Hoxb1null mice show an auditory threshold shift, most probably because of the outer hair cells loss. In addition, they demonstrate the loss of cholinergic olivocochlear efferent innervation onto developing inner hair cells from the Hoxb1null mouse. These data nicely confirm our anatomical and morphological data on the loss of cholinergic efferents, previously published.

Genetic types of deafness can lead to non-syndromic or syndromic forms of human deafness. Therefore, the detailed analysis of hearing deficits and the identification of subpopulations, subcircuits and genes involved in the assembly of a normal auditory system are a prerequisite to envision personalized future therapies.

Because hearing loss greatly impairs quality of life and leads to a substantial economical cost, understanding the contribution of the different neuronal structures and circuits on the sound encoding process becomes a high priority for a personalized therapeutical approach. However, studies related to understanding the formation of the auditory system have lagged behind other sensory systems, most probably because of the high complexity of the auditory system. We are in the process of dissecting the auditory circuit into specific subcircuits and identifying neuronal populations contributing to these circuits. In addition, we plan to identify genes involved in different forms of deafness that should foster the development of new algorithms for hearing aids and cochlear implants, together with genetic screening and pharmacological-based therapies for patients.

Finally, there is a huge interest in the hindbrain field to generate rhombomere-specific transgenic lines. This, mainly because the origin and function of distinct hindbrain subpopulations are poorly understood, differently from the spinal cord field in which more data are available. Thus, the transgenic mice produced as part of this project will be made available to the general scientific community. The coordinator will attend different scientific meetings to update the scientific community on the project advancement. Last but not least, being a basic research type of proposal, the ultimate goal of exploiting the results obtained from this project is also to publish in high-profile journals and thus to reach a wide audience of biologists, neurophysiologists and clinicians.

Publications:
1. Di Bonito, M., Boulland, J.L., Krezel W., Setti E., Studer, M.*, Glover, J.C.* Loss of projections, functional compensation and residual deficits in the mammalian vestibulospinal system of Hoxb1-deficient mice; eNeuro, 2015,Dec 26;2(6). doi: 10.1523/ENEURO.0096-15.2015.
2. Di Bonito M., Studer M., Puelles L. (2017) Nuclear derivatives and axonal projections originating from rhombomere 4 in the mouse hindbrain. Brain structure and functions.2017 May 3. doi: 10.1007/s00429-017-1416-0
3. Di Bonito M. and Studer M. (2017) Cellular and Molecular Underpinnings of Neuronal Assembly in the Central Auditory System during Mouse Development. Frontiers in Neuronal Circuits : 11 :18 doi :10.3389/fncir.2017.00018

Poster presentations:
1. Society of Neuroscience USA 2016. San Diego, CA, November 12-16, 2016. Di Bonito M., Setti E., Puelles L., Studer M. Anatomical, Molecular and Functional Characterization of Rhombomere 4-derived Sensorimotor Subcircuits.
2. 21st Biennial Meeting of the International Society for Developmental Neuroscience, ISDN 2016, Antibes-Juan Les Pins, France May 11-14, 2016. Di Bonito M., Setti E., Studer M. Characterization of rhombomere 4-derived sensorimotor systems in the brainstem.

Hearing relies on precisely organized circuits to faithfully transfer the acoustic stimulation of our environment from the cochlea to the brain. Hearing loss and hereditary deafness are major health issues resulting from noise, aging, disease, and genetics. Sensorineural hearing loss originates from malformations in the inner ear structures (i.e., cochlea) and/or abnormal transmission of impulses along the auditory nerve. The mechanisms underlying this type of deafness are still not well elucidated.

The auditory system, formed by sensory and motor neurons, is assembled during development into precise subcircuits and depends on the spatially and temporally ordered sequence of specification, migration and connectivity. Alterations of one of these processes will lead to abnormal circuit formation and are at the basis of different types of hearing impairments. We previously showed that several auditory nuclei along the ascending hearing pathway and two sensory-motor feedback subcircuits originate in rhombomere 4 (r4), a distinct region of the developing brainstem. In the absence of Hoxb1, a key gene in imposing r4 identity and cell-type specification, mice have severe hearing impairments. Mutant embryos abnormally develop some auditory sensory nuclei involved in the transmission of the sound and fail to generate efferent motor neurons innervating the cochlea. Young mutant animals show severe degeneration of cochlear hair cells leading to an abnormal cochlear amplification process in which low-amplitude sounds are not perceived. Interestingly, this phenotype well reproduces the hearing defects of patients with a missense mutation in the human HOXB1, making the mouse model an invaluable tool to dissect mechanisms underlying sensorineural hearing impairments.

In this proposal, we aim to contribute to the identification of neuronal populations and subcircuits involved in this type of hearing defect by combining genetic, molecular, cellular and electrophysiological approaches. First, we will take advantage of the intersectional genetic approach, which uses a combination of Cre-loxP and Flp-FRT recombinases in the mouse, to functionally characterize the individual cell populations and subcircuits required for proper assembly of the auditory system. We will combine our recently generated r4-Flippase line with subtype-specific Cre-recombinase lines of different dorsal sensory and ventral motor domains to genetically identify the origin of distinct auditory subpopulations within r4 and follow them in time and space.

This genetic strategy will then be transferred to a Hoxb1 mutant background so that auditory-specific subpopulations, either sensory or motor, can be selectively altered and the anatomical and electrophysiological properties of the ascending pathway and the cochlea can be investigated from embryonic to adult stages. This approach will not only dissect the mechanisms at the origin of the auditory impairments previously described in constitutive Hoxb1 mouse mutants and patients carrying mutations in the HOXB1 gene, but also contribute to our understanding on how different subtypes that co-operate within the auditory system, acquire distinct functions and, ultimately, how their alterations affect diverse aspects of hearing.

Finally, the intersectional genetic strategy will enable to capture selected neuronal subpopulations and to identify, through genomic approaches, novel molecular players involved in auditory circuit assembly and affected in sound impairments. The overall aim of this proposal is thus to contribute to our understanding of the mechanisms involved in hearing deficit in human diseases and to identify new genes required in the assembly of the auditory system, with the ultimate goal to improve the development of novel therapies.