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

Abstract

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.

Fig. 1

The evolution of the basilar papilla/organ of Corti is depicted in this scheme. A basilar papilla at the orifice of the lagenar recess harboring the gravistatic lagenar organ is a common feature of all tetrapods. The most parsimonious explaination would be that a single evolutionary event has led to the formation of a lagena, a lagenar recess and the basilar papilla (left). Data on the sarcopterygian fish, Latimeria, suggests that this evolutionary event dates back to the aquatic ancestor of tetrapods. Monotremes have retained the original configuration of tetrapods and have a lagenar recess with a lagena sensory epithelium (middle). However, while the organ of Corti of monotremes is in the same position as the basilar papilla of tetrapods, it has evolved the cellular architecture of the organ of Corti with inner and outer hair cells. The coiling of the cochlear duct and extension of the organ of Corti throughout the cochlear duct in modern mammals (marsupials, eutherians) is somehow related to the evolutionary and developmental loss of the lagena (right). Adopted from (Jorgensen and Locket, 1995; Pauley et al., 2003)

Fig. 2. Lack of Foxg1 results in a shortened and wider organ of Corti

Absence of Foxg1 results at E18.5 (A,B,C) in cellular disorganization of the organ of Corti. Multiple rows of inner and outer hair cells form in the apex (B,B′) with near normal organization in the base (A,A′). Despite the presence of multiple rows of hair cells, their innervation is severely disrupted with overshooting fibers towards the lateral wall of the cochlea (A,B). The cross section through the apex and the base of the cochlea of the Foxg1 null ear show near normal orientation of inner and outer hair cells in base (A″), but the apex displays ten rows of outer hair cells with multiple supporting cells (B″). The SEM demonstrates apical specializations of the multiple rows of disorganized hair cells (C). The polarity of hair cells is distorted in the apex of the Foxg1 null cochlea (C) indicating that Foxg1 is not only needed for convergent extension movements but also plays a role in planar polarity formation. The apex of Foxg1 null mice resembles the spiny anteater (a monotreme) cochlea. The extant monotremes (spiny anteater and platypus) are the only mammals that have multiple rows of hair cells in a shortened cochlea (D). GER, greater epithelial ridge; IHC, inner hair cells; OHC, outer hair cells; TM, tectorial membrane; SC, supporting cells. C, modified after (Pauley et al., 2006); D, modified after (Ladhams and Pickles, 1996). Bar indicates 100 um in A,A′,B,B′ and 10 um in A″,B″,C,D.

Fig. 3. Absence of Neurog1 and Neurod1 leads to truncation of cochlear elongation

Deletion of Neurog1 stunts cochlear growth (A), results in multiple rows of inner and outer hair cells near the apex (A′) and the formation of hair cells in the GER and ductus reunions (A′, A″). The apical tip is characterized by 1–2 rows of only inner hair cells and complete absence of outer hair cells (A‴). Note the near complete loss of saccular hair cells (S in A″, insert in A‴). Floxed Neurod1 conditional knockout (CKO) using Pax2-cre also results in a shorter cochlea (B) and the formation of two rows of inner and four or more rows of outer hair cells in apical half of the organ of Corti (C,D). Neurod1 CKO mice are viable and studying the postnatal hair cells reveals developmental deviations with ectopic inner hair cell formation (C,D) in positions that are topographically equivalent to outer hair cells. Some Deiter’s cells show pillar cell like differentiation (C′). As with Neurog1 null mice (A‴), Neurod1 CKO mice show only inner hair cells in an irregular row in the apical tip (E,E′) that receive an unusual pattern of innervation of the greatly diminished spiral neurons (E). DR, ductus reunions; GER, greater epithelial ridge; OC, organ of Corti; S, saccule; U, utricle. Bar indicates 100 um except in C–E′ where it indicates 10 um.

Fig. 4. An organ of Corti consisting of undifferentiated cells develops in Atoh1 null mice

Absence of the bHLH gene Atoh1 results in agenesis of hair cells. However, using either the hair cell marker Bdnf (A,B) or the LacZ reporter knocked into the Atoh1 locus (C) reveals expression of these markers suggesting some degree of hair cell precursor specification in mice lacking Atoh1 (B,C). Wild type mice show upregulation of Bdnf in all hair cells throughout the cochlea (A, A′, A″). In contrast, there is no expression of Bdnf in the base of the cochlea (B) whereas it is expanded in the apex without any recognizable rows of inner and outer hair cells (B, B′). Sections show distribution of the Bdnf marker throughout all cells in the undifferentiated organ of Corti (B″). The LacZ reporter inserted to replace the coding frame of the Atoh1 gene shows expression along the entire length of the organ of Corti (C). However, this expression is reduced to 1–2 irregular rows of cells in the base (C′) and much wider in the apex (C). Radial sections through the organ of Corti show the β-galactosidase reaction product throughout the height of the organ of Corti (C″). Occasionally, cells in the correct topology of outer hair cells may be found (arrows in C″). IHC, inner hair cells; OHC, outer hair cells. Bar indicates 100 um except in A″, B″, C″ where it indicates 10 um.

Fig. 5. Lack of Neurod1 results in formation of hair cells in the remaining ganglia

Mice with conditional deletion of Neurod1(Pax2-cre::Neurod1 ^f/f; A–C) have a reduction in innervation as shown with anti-tubulin (A, green) but also show an unusual distribution of Myo7a positive cells in the remaining ganglia in addition to the hair cells of the sensory epithelia. These cells persist in adult animals and form hair cell-like tufts extending into multiple large and small vesicles that form inside the ganglia (C,D). Pax2-cre mediated Rosa 26 LacZ expression (B, C′) shows that neurons and hair cells have mostly β-galactosidase positive nuclei. This level of co-localization suggests that hair cells and neurons of ganglia derive from the otocyst, and that hair cell may derive from neuronal precursors that alter their fate in the absence of Neurod1 into hair cells. AC, anterior crista; HC, horizontal crista; U, utricle; V, intraganglionic vesicle; Vgl, vestibular ganglion. Bar indicates 100 um except in B′, which indicates 10 um.

Fig. 6

This scheme displays the time of cell cycle exit and some of the transcription factors known or suspected to play a role in organ of Corti and hair cell development. Note that Sox2 and Gata3 are expressed throughout the organ of Corti prior to, during and after cell cycle exit. They could promote cell cycle progression but are unlikely to be causal for either defining alone the organ of Corti or expression of hair cell specific bHLH genes. In contrast to the apex to base progression of the cell cycle exit, expression of bHLH genes is from upper middle turn (‘base’) to apex. Indeed, expression of bHLH genes reaches the first hair cells that exit the cell cycle in the apex around E12.5 only around birth. This delay indicates that Atoh1 (and other bHLH transcription factors) play no role in cell cycle exit but act as differentiation factors in the organ of Corti. When exactly first expressions occur will depend on the techniques used. Some factors (Neurod1) have early expression in general followed much later by specific expressions. Modified after (Fritzsch et al., 2010a; Karis et al., 2001; Matei et al., 2005; Nichols et al., 2008; Ruben, 1967).