Here we describe a mouse line in which all pendrin is derived from dox-induced expression of an Slc26a4 cDNA transgene. Our data reveal a developmental period, E16.5 to P2, during which expression of Slc26a4 is required for acquisition of normal hearing (Figure ), although the EP did not reach normal levels (Figure A). This may suggest that pendrin is required over a longer period for the development of a normal EP. It could also indicate that Tg[E];Tg[R];Slc26a4Δ/Δ mice are unable to acquire a normal EP even in the presence of continuous dox exposure. Continued expression of pendrin through P6 increased the EP, but not to normal levels. The onsets of the critical intervals for dox initiation (E16.5) and discontinuation (E15.5) differ by 1 day. This likely reflects the slower kinetics associated with discontinuation of dox and pendrin turnover. The results of the discontinuation and initiation models are thus consistent with each other and collectively suggest that pendrin is required during a limited period at or shortly after E16.5 for development of normal hearing.
The critical time interval for pendrin expression corresponds to the period during which the cochlea grows rapidly (20
). The transiency of this critical time interval for pendrin expression could reflect a temporary period of sensitivity of inner ear development to disruptions of endolymph pH or ionic homeostasis. It is also possible that pendrin mediates exchange of an anion, base, or both that is only temporarily required for normal inner ear physiology and development. I–
is a known in vitro exchange substrate of pendrin (25
), and its concentration probably rises soon following the onset of type 2 deiodinase activity in the late embryonic cochlea (26
). Cochlear concentrations and mechanism of I–
homeostasis are unknown but, if there were not a mechanism to disperse I–
, an excess could conceivably exert a toxic effect on cells within the cochlea, inhibit deiodinase reaction(s) leading to cochlear hypothyroidism (27
), or produce a combination of these effects. Finally, the dependence of renal Na+
reabsorption on luminal HCO3–
secretion and alkalinization by pendrin (28
) raises the possibility of an analogous role in embryonic endolymph. Although K+
is the major osmolyte in mature endolymph, the composition of embryonic endolymph is unknown and Na+
may be an important developmental osmolyte. A decrease in Na+
reabsorption thus might be poorly tolerated, leading to increased osmotic pressure, scala media acidification and expansion, and EVA.
Pendrin is not required, or is required only at undetectable low levels, for acquisition of normal hearing in a normally formed mature inner ear. This raises the possibility that replacement or upregulation of pendrin in a mature ear might not reverse or prevent hearing loss caused by SLC26A4 mutations in human subjects. However, we have not yet determined whether the low EP associated with normal hearing in Tg[E];Tg[R];Slc26a4Δ/Δ DP6 ears leads to later-onset hearing loss. If so, upregulation of pendrin may still be a logical therapeutic objective.
One advantage of inducible Slc26a4
expression is the opportunity to distinguish early primary pathogenetic events from later secondary effects. We were able to distinguish functional consequences associated with loss of pendrin expression in a mature cochlea versus secondary effects of the severe structural malformations associated with complete pendrin deficiency. Both Tg[E];Tg[R];Slc26a4Δ/Δ
IE18.5 (dox i
nitiated at E18.5
) and DE17.5 (dox d
iscontinued at E17.5
) mice had severe to profound hearing loss, yet scala media enlargement was present in IE18.5 mice only during embryonic development (Figure C and ref. 20
) and was not observed in DE17.5 mice (Figure B). Therefore scala media expansion is unlikely to be a sole direct cause of loss of EP and hearing.
We observed scala media acidification in both Tg[E];Tg[R];Slc26a4Δ/Δ
IE18.5 and DE17.5 mice, confirming that cochlear endolymph acidification may indeed be an early pathogenetic event ultimately leading to reduction of the EP and hearing in Slc26a4
-deficient ears. The reduction of EP might be mediated by functional degradation of the normally tight permeability barrier of the basal stria vascularis that is required for maintenance of the EP (29
). However, our observation of normal overall levels of Kcnj10 immunoreactivity (Figure , A and C) does not rule out a decrease of functional Kcnj10 at the plasma membrane of intermediate cells of the stria vascularis that generate the EP. The loss of the EP and hearing in Slc26a4
-deficient mice could result from either of these or other mechanisms that disrupt the function of the stria vascularis and lateral wall of the cochlea.
The results of this study and others (16
) suggest that preserving or supporting strial function and thereby preventing hair cell loss could be a primary target of strategies to prevent or retard hearing loss caused by SLC26A4
mutations. Prevention of scala media acidification could be another goal.
One difference between Slc26a4Δ/Δ
mice and Tg[E];Tg[R];Slc26a4Δ/Δ
IE18.5 or DE17.5 mice is the lack of endolymphatic hydrops (scala media expansion with distention of Reissner membrane) in the adult cochleae of the inducible expression models. The reported time line of endolymphatic enlargement in Slc26a4Δ/Δ
) suggests that Tg[E];Tg[R];Slc26a4Δ/Δ
IE18.5 cochleae also develop endolymphatic hydrops with distention of Reissner membrane until E18.5. The distended Reissner membrane subsequently collapses onto the tectorial membrane by 1 month of age (Figure ) upon reversal of endolymphatic hydrops with induction of pendrin expression at E18.5. In contrast, Tg[E];Tg[R];Slc26a4Δ/Δ
DE17.5 mice probably never develop endolymphatic hydrops, since DE16.5 inner ears show no endolymphatic enlargement.
Induction of Slc26a4 expression at E18.5 did not rescue the ability to express pendrin expression in the outer sulcus of Tg[E];Tg[R];Slc26a4Δ/Δ IE18.5 cochleae (Figure ). This finding likely reflects a developmental disruption or retardation of nonsensory epithelial cell function in the outer sulcus or spiral prominence (i.e., the cells that express pendrin).
The etiopathogenesis of incremental and progressive hearing loss in humans with SLC26A4 mutations was not addressed by our study. Elucidation of the factors underlying hearing loss stability is important, since stabilization of hearing in humans with EVA is an important therapeutic goal. Tg[E];Tg[R];Slc26a4Δ/Δ mice may be used to explore these issues, since the IE18.5 and DE17.5 paradigms result in residual hearing. The hearing loss observed with these paradigms, particularly the asymmetry and unilaterality associated with the DE19.5, DP0, and DP1 paradigms, is remarkable for its similarity to some human EVA phenotypes. Characterization of the natural history of hearing loss associated with these dox administration paradigms in Tg[E];Tg[R];Slc26a4Δ/Δ mice will be needed to fully explore and exploit this model of human hearing loss associated with EVA.