Benjamin Kopecky

Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti.

Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell death. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), the remaining cells of the organ of Corti are transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, except for the apex and few fibers in the base. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.

A novel Atoh1 "self-terminating" mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability.

Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear.

Ear development requires interactions of transcription factors for proliferation and differentiation. The proto‐oncogene N‐Myc is a member of the Myc family that regulates proliferation. To investigate the function of N‐Myc, we conditionally knocked out N‐Myc in the ear using Tg(Pax2‐Cre) and Foxg1KiCre. N‐Myc CKOs had reduced growth of the ear, abnormal morphology including fused sensory epithelia, disrupted histology, and disorganized neuronal innervation. Using Thin‐Sheet Laser Imaging Microscopy (TSLIM), 3D reconstruction and quantification of the cochlea revealed a greater than 50% size reduction. Immunochemistry and in situ hybridization showed a gravistatic organ‐cochlear fusion and a “circularized” apex with no clear inner and outer hair cells. Furthermore, the abnormally developed cochlea had cross innervation from the vestibular ganglion near the basal tip. These findings are put in the context of the possible functional relationship of N‐Myc with a number of other cell proliferative and fate determining genes during ear development. Developmental Dynamics 240:1373–1390, 2011.

Evolution and development of the tetrapod auditory system: an organ of Corti-centric perspective

The tetrapod auditory system transmits sound through the outer and middle ear to the organ of Corti or other sound pressure receivers of the inner ear where specialized hair cells translate vibrations of the basilar membrane into electrical potential changes that are conducted by the spiral ganglion neurons to the auditory nuclei. In other systems, notably the vertebrate limb, a detailed connection between the evolutionary variations in adaptive morphology and the underlying alterations in the genetic basis of development has been partially elucidated. In this review, we attempt to correlate evolutionary and partially characterized molecular data into a cohesive perspective of the evolution of the mammalian organ of Corti out of the tetrapod basilar papilla. We propose a stepwise, molecularly partially characterized transformation of the ancestral, vestibular developmental program of the vertebrate ear. This review provides a framework to decipher both discrete steps in development and the evolution of unique functional adaptations of the auditory system. The combined analysis of evolution and development establishes a powerful cross‐correlation where conclusions derived from either approach become more meaningful in a larger context which is not possible through exclusively evolution or development centered perspectives. Selection may explain the survival of the fittest auditory system, but only developmental genetics can explain the arrival of the fittest auditory system. [Modified after (Wagner 2011)]

Dissecting the molecular basis of organ of Corti development: Where are we now?

This review summarizes recent progress in our understanding of the molecular basis of cochlear duct growth, specification of the organ of Corti, and differentiation of the different types of hair cells. Studies of multiple mutations suggest that developing hair cells are involved in stretching the organ of Corti through convergent extension movements. However, Atoh1 null mutants have only undifferentiated and dying organ of Corti precursors but show a near normal extension of the cochlear duct, implying that organ of Corti precursor cells can equally drive this process. Some factors influence cochlear duct growth by regulating the cell cycle and proliferation. Shortened cell cycle and premature cell cycle exit can lead to a shorter organ of Corti with multiple rows of hair cells (e.g., Foxg1 null mice). Other genes affect the initial formation of a cochlear duct with or without affecting the organ of Corti. Such observations are consistent with evolutionary data that suggest some developmental uncoupling of cochlear duct from organ of Corti formation. Positioning the organ of Corti requires multiple genes expressed in the organ of Corti and the flanking region. Several candidate factors have emerged but how they cooperate to specify the organ of Corti and the topology of hair cells remains unclear. Atoh1 is required for differentiation of all hair cells, but regulation of inner versus outer hair cell differentiation is still unidentified. In summary, the emerging molecular complexity of organ of Corti development demands further study before a rational approach towards regeneration of unique types of hair cells in specific positions is possible.

Expression of Neurog1 instead of Atoh1 can partially rescue organ of Corti cell survival.

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons.

Understanding the evolution and development of neurosensory transcription factors of the ear to enhance therapeutic translation

Reconstructing a functional organ of Corti is the ultimate target towards curing hearing loss. Despite the impressive technical gains made over the last few years, many complications remain ahead for the two main restoration avenues: in vitro transformation of pluripotent cells into hair cell-like cells and adenovirus-mediated gene therapy. Most notably, both approaches require a more complete understanding of the molecular networks that ensure specific cell types form in the correct places to allow proper function of the restored organ of Corti. Important to this understanding are the basic helix-loop-helix (bHLH) transcription factors (TFs) that are highly diverse and serve to increase functional complexity but their evolutionary implementation in the inner ear neurosensory development is less conspicuous. To this end, we review the evolutionary and developmentally dynamic interactions of the three bHLH TFs that have been identified as the main players in neurosensory evolution and development, Neurog1, Neurod1 and Atoh1. These three TFs belong to the neurogenin/atonal family and evolved from a molecular precursor that likely regulated single sensory cell development in the ectoderm of metazoan ancestors but are now also expressed in other parts of the body, including the brain. They interact extensively via intracellular and intercellular cross-regulation to establish the two main neurosensory cell types of the ear, the hair cells and sensory neurons. Furthermore, the level and duration of their expression affect the specification of hair cell subtypes (inner hair cells vs. outer hair cells). We propose that appropriate manipulation of these TFs through their characterized binding sites may offer a solution by itself, or in conjunction with the two other approaches currently pursued by others, to restore the organ of Corti.

Regeneration of Hair Cells: Making Sense of All the Noise.

Hearing loss affects hundreds of millions of people worldwide by dampening or cutting off their auditory connection to the world. Current treatments for sensorineural hearing loss (SNHL) with cochlear implants are not perfect, leaving regenerative medicine as the logical avenue to a perfect cure. Multiple routes to regeneration of damaged hair cells have been proposed and are actively pursued. Each route not only requires a keen understanding of the molecular basis of ear development but also faces the practical limitations of stem cell regulation in the delicate inner ear where topology of cell distribution is essential. Improvements in our molecular understanding of the minimal essential genes necessary for hair cell formation and recent advances in stem cell manipulation, such as seen with inducible pluripotent stem cells (iPSCs) and epidermal neural crest stem cells (EPI-NCSCs), have opened new possibilities to advance research in translational stem cell therapies for individuals with hearing loss. Despite this, more detailed network maps of gene expression are needed, including an appreciation for the roles of microRNAs (miRs), key regulators of transcriptional gene networks. To harness the true potential of stem cells for hair cell regeneration, basic science and clinical medicine must work together to expedite the transition from bench to bedside by elucidating the full mechanisms of inner ear hair cell development, including a focus on the role of miRs, and adapting this knowledge safely and efficiently to stem cell technologies.

Scanning thin-sheet laser imaging microscopy elucidates details on mouse ear development

Background: The mammalian inner ear is transformed from a flat placode into a three‐dimensional (3D) structure with six sensory epithelia that allow for the perception of sound and both linear and angular acceleration. While hearing and balance problems are typically considered to be adult onset diseases, they may arise as a developmental perturbation to the developing ear. Future prevention of hearing or balance loss requires an understanding of how closely genetic mutations in model organisms reflect the human case, necessitating an objective multidimensional comparison of mouse ears with human ears that have comparable mutations in the same gene. Results: Here, we present improved 3D analyses of normal murine ears during embryonic development using optical sections obtained through Thin‐Sheet Laser Imaging Microscopy. We chronicle the transformation of an undifferentiated otic vesicle between mouse embryonic day 11.5 to a fully differentiated inner ear at postnatal day 15. Conclusions: Our analysis of ear development provides new insights into ear development, enables unique perspectives into the complex development of the ear, and allows for the first full quantification of volumetric and linear aspects of ear growth. Our data provide the framework for future analysis of mutant phenotypes that are currently under‐appreciated using only two dimensional renderings. Developmental Dynamics 241:465–480, 2012.

Correct Timing of Proliferation and Differentiation is Necessary for Normal Inner Ear Development and Auditory Hair Cell Viability

Background: Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2‐Cre N‐Myc conditional knockout (CKO) mice highlighted the need of N‐Myc for proper neurosensory development and possible redundancy with L‐Myc. The late‐onset hair cell death in the absence of early N‐Myc expression could be due to mis‐regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1. Results: Pax2‐Cre N‐Myc L‐Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation result in morphologically abnormal ears. In particular, the organ of Corti apex is re‐patterned into a vestibular‐like organization and the base is truncated and fused with the saccule. Conclusions: These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix‐Loop‐Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development. Developmental Dynamics 242:132–147, 2013.