The data presented here demonstrate previously unreported roles for HH signaling in the development and function of the mammalian cochlea. All of the structures within the membraneous labyrinth of the inner ear are derived from the epithelial cell-lined otocyst. The factors that specify some regions of the otocyst to develop as prosensory cells, while other regions develop as non-sensory, remain largely unknown. This is a particularly intriguing question for the mammalian cochlear duct. Normally, only a limited percentage of cells within the duct become prosensory cells, however several recent studies have demonstrated that cells in other regions of the duct possess the ability to develop as sensory cell types, including hair cells (Zheng and Gao, 2000
; Kawamoto et al., 2003
; this study) and supporting cells (Zheng and Gao, 2000
; Kawamoto et al., 2003
; Woods et al., 2004
). While activation of the Notch pathway and expression of Sox2 have been implicated as important regulators of prosensory formation, the factors that regulate either of these events are unknown. The results presented here suggest that, at least for the cochlear duct, HH signaling plays an important role in repressing prosensory formation. The presence of an asymmetric source of Shh originating in the medially located spiral ganglion is consistent with a higher level of HH signaling in the medially located Kölliker’s organ. Reduction of HH signaling either through the exclusive presence of the repressor form of Gli3 or the addition of KAAD-cyclopamine or HIP leads to a loss of HH-mediated repression, an increase in the size of the endogenous prosensory domain and the spontaneous formation of ectopic prosensory domains within Kölliker’s organ. The formation of these ectopic prosensory domains is dependent on Notch signaling, as significantly fewer ectopic hair cell patches are observed when Notch signaling is blocked by DAPT treatment. This result is consistent with the recent demonstration that prosensory formation is dependent on activation of Notch, presumably by Jagged1 (Kiernan et al., 2006
). The result also suggests that the SHH and Notch signaling pathways normally interact antagonistically to define the position and size of the prosensory domain in the developing cochlea. Antagonistic interactions between SHH and Notch signaling have been reported in some systems (Nicolas et al., 2003
); however, it is also possible that HH signaling influences prosensory formation upstream from Jagged1/Notch activation. It is possible that the ectopic sensory patches arise as a result of migration of prosensory cells from the endogenous prosensory domain. However, while this possibility cannot be completely discounted, the similarities in the patterns of ectopic Sox2 expression in KAAD-cyclopamine-treated explants after 48 hours and ectopic hair cell formation in KAAD-cyclopamine-treated explants after 6 days suggests that ectopic prosensory cells arise spontaneously within Kölliker’s organ.
The formation of ectopic hair cells in the presence of reduced HH activity may have intriguing implications regarding the evolution of the mammalian cochlea. In placental mammals, the sensory epithelium comprises a relatively small percentage of the cochlear duct by comparison with other vertebrate classes or in non-placental mammals. While the basis for this change has not been determined, it has been suggested that the total size of the sensory epithelium may have become reduced in response to selective pressures related to the perception of high frequencies (Rubel, 1978
). Since, as mentioned previously, the cells of Kölliker’s organ can form HCs or supporting cells, it seems possible that some regions of Kölliker’s organ may have developed as sensory cells in an ancestral cochlear duct. Therefore, HH signaling may have evolved to repress prosensory formation within this region.
Based on stereociliary bundle morphology and the presence of calyceal neuronal terminals, the ectopic HCs in Gli3Δ699/Δ699 mutant cochleae are vestibular in nature. Since the stereociliary bundles on the endogenous cochlear HCs appear normal in these mutants, the vestibular phenotype of the ectopic HCs is unlikely to be due to a requirement for full-length Gli3 or HH signaling for cochlear HC or stereociliary bundle development. Rather, since mammalian cochlear hair cells are more derived, the ectopic hair cells may be differentiating along an ancestral developmental pathway. As a result of their location, the ectopic cells in Gli3Δ699/Δ699 mutants presumably do not receive all the instructive signals necessary to form the unique mammalian cochlear HC types, and thus default to an earlier, ancestral form. It would be interesting to compare the role of hedgehog signaling in development of the auditory sensory epithelia in other vertebrates to see if this is indeed the case.
The presence of adjacent supporting cells and neuronal terminals on these ectopic HCs suggests that these cells are probably functional. Among the Gli3Δ699/Δ699
cochleae examined, variable degrees of cellular mis-patterning within the organ of Corti and generation of ectopic hair cells within Kölliker’s organ were observed. Since overproduction of hair cells can lead to hearing loss (Chen and Segil, 1999
), the presence and innervation of both additional rows of hair cells within the OC and ectopic hair cells in Kölliker’s organ likely contributes to disruption of sensorineural function. This result is consistent with the variable high frequency hearing loss observed in many PHS subjects.
Many PHS patients show evidence of auditory defects that are consistent with a shortening of the cochlear duct. Low frequency hearing loss, indicative of defects in the apical portion of the cochlea, was observed in all PHS subjects with auditory deficits. While neonatal lethality prevented any assessment of auditory function in Gli3Δ699/Δ699
mice, direct measurement of cochlear length demonstrated a significant shortening of the cochlea relative to WT. The Gli3Δ699/Δ699
phenotype of a shortened cochlea, but unaffected vestibular structures, is consistent with earlier work showing that Shh
signaling is required for development of the ventral (cochlear) but not the dorsal (vestibular) portion of the inner ear (Riccomagno et al., 2002
; Bok et al., 2005
; Riccomagno et al., 2005
; Bok et al., 2007
). GLI activator function is apparently required for transducing positive Shh
signaling in this context for the proper outgrowth of the cochlear duct. However, we have not observed shortening of the cochlear duct in magnetic resonance images of the inner ears of a PHS patient (unpublished observation).
In summary, we have shown here that HH signaling is necessary for proper development of the cochlea and sensory epithelium in mice, and for auditory function in humans. The results demonstrate a new role for HH signaling as a repressor of prosensory fate within the cochlear duct. The results also suggest that specification of prosensory domains within the inner ear does not represent a purely inductive process, and that at least some regions of the cochlear duct that normally develop as non-sensory, may possess an innate ability to adopt a prosensory fate. These results have intriguing implications in terms of understanding developmental patterning of the otocyst and the potential for spontaneous hair cell formation in the adult inner ear.