Elizabeth C. Driver

Hedgehog Signaling Regulates Sensory Cell Formation and Auditory Function in Mice and Humans

Auditory perception is mediated through a finite number of mechanosensory hair cells located in a specialized sensory epithelium within the inner ear. The formation of the appropriate number of hair cells and the location of those cells is crucial for normal auditory function. However, the factors that regulate the formation of this epithelium remain poorly understood. Truncating mutations in the transcription factor GLI3, a downstream effector of the Hedgehog (HH) pathway, lead to a partial loss of HH signaling and cause Pallister-Hall syndrome (PHS). Here, we report that cochleae from a mouse model of PHS (Gli3Δ699), which produces only the truncated, repressor form of GLI3, have a variably penetrant phenotype that includes an increase in the size of the sensory epithelium and the development of large ectopic sensory patches in Köllikers organ (KO). Consistent with the mouse model, some PHS individuals exhibit hearing loss across a broad range of frequencies. Moreover, inhibition of HH signaling in vitro results in an increase in the size of the prosensory domain, a precursor population that gives rise to the sensory epithelium, whereas treatment with Sonic hedgehog (SHH) inhibits prosensory formation. Finally, we demonstrate that HH signaling within the cochlea regulates expression of prosensory markers and that the effects of HH in KO are dependent on activation of Notch, an inducer of prosensory fate. These results suggest that HH signaling plays a key role in the specification, size, and location of the prosensory domain, and therefore of hair cells, within the cochlea.

Specification of cell fate in the mammalian cochlea

Mammalian auditory sensation is mediated by the organ of Corti, a specialized sensory epithelium found in the cochlea of the inner ear. Proper auditory function requires that the many different cell types found in the sensory epithelium be precisely ordered within an exquisitely patterned cellular mosaic. The development of this mosaic depends on a series of cell fate decisions that transform the initially nearly uniform cochlear epithelium into the complex structure of the mature organ of Corti. The prosensory domain, which contains the progenitors of both the mechanosensory hair cells and their associated supporting cells, first becomes distinct from both the neural and the nonsensory domains. Further cell fate decisions subdivide prosensory cells into populations of inner and outer hair cells, and several different types of supporting cells. A number of different signaling pathways and transcription factors are known to be necessary for these developmental processes; in this review, we will summarize these results with an emphasis on recent findings.

Regulation of cell fate and patterning in the developing mammalian cochlea.

Purpose of reviewA significant proportion of hearing loss and deafness is caused by defects in the structure or function of cells within the organ of Corti. Identification of the molecular factors that regulate the development of this structure should provide valuable insights regarding inner ear formation and the signaling pathways that underlie congenital auditory deficits. In addition, targeted modulation of these same factors could be developed as therapies for hair cell regeneration. Recent findingsResults from experiments using transgenic and mutant mice, as well as in-vitro techniques, have identified genes and signaling pathways that are required to either specify unique auditory cell types, such as hair cells or supporting cells, or to generate the highly ordered cellular pattern that is characteristic for the organ of Corti. In particular, the hedgehog and fibroblast growth factor signaling pathways modulate the formation of the progenitor cells that will give rise to the organ of Corti. SRY-box containing gene 2, a transcription factor that is required for the formation of the cochlear progenitor cell population, has paradoxically been shown to also act as an inhibitor of hair cell development. Finally, the motor protein myosin II regulates extension of the organ of Corti and the alignment of hair cells and supporting cells into ordered rows. SummaryA better understanding of the signaling pathways that direct different aspects of cochlear development, such as specific of cell fates or cellular patterning, offers the potential to identify new pathways or molecules that could be targeted for therapeutic interventions.

Focusing forward genetics: a tripartite ENU screen for neurodevelopmental mutations in the mouse.

The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.

Transfection of Mouse Cochlear Explants by Electroporation

The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non‐sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5. Curr. Protoc. Neurosci. 51:4.34.1‐4.34.10.

Cell migration, intercalation and growth regulate mammalian cochlear extension

Developmental remodeling of the sensory epithelium of the cochlea is required for the formation of an elongated, tonotopically organized auditory organ, but the cellular processes that mediate these events are largely unknown. We used both morphological assessments of cellular rearrangements and time-lapse imaging to visualize cochlear remodeling in mouse. Analysis of cell redistribution showed that the cochlea extends through a combination of radial intercalation and cell growth. Live imaging demonstrated that concomitant cellular intercalation results in a brief period of epithelial convergence, although subsequent changes in cell size lead to medial-lateral spreading. Supporting cells, which retain contact with the basement membrane, exhibit biased protrusive activity and directed movement along the axis of extension. By contrast, hair cells lose contact with the basement membrane, but contribute to continued outgrowth through increased cell size. Regulation of cellular protrusions, movement and intercalation within the cochlea all require myosin II. These results establish, for the first time, many of the cellular processes that drive the distribution of sensory cells along the tonotopic axis of the cochlea. Summary: Myosin II plays multiple roles in the cell morphology changes and cellular rearrangements, including radial intercalation, that drive remodeling of the prosensory domain during development of the mouse cochlea.