Evolution of Endolymph Secretion and Endolymphatic Potential Generation in the Vertebrate Inner Ear.

The ear of extant vertebrates reflects multiple independent evolutionary trajectories. Examples include the middle ear or the unique specializations of the mammalian cochlea. Another striking difference between vertebrate inner ears concerns the differences in the magnitude of the endolymphatic potential. This differs both between the vestibular and auditory part of the inner ear as well as between the auditory periphery in different vertebrates. Here we provide a comparison of the cellular and molecular mechanisms in different endorgans across vertebrates. We begin with the lateral line and vestibular systems, as they likely represent plesiomorphic conditions, then review the situation in different vertebrate auditory endorgans. All three systems harbor hair cells bathed in a high (K+) environment. Superficial lateral line neuromasts are bathed in an electrogenically maintained high (K+) microenvironment provided by the complex gelatinous cupula. This is associated with a positive endocupular potential. Whether this is a special or a universal feature of lateral line and possibly vestibular cupulae remains to be discovered. The vestibular system represents a closed system with an endolymph that is characterized by an enhanced (K+) relative to the perilymph. Yet only in land vertebrates does (K+) exceed (Na+). The endolymphatic potential ranges from +1 to +11 mV, albeit we note intriguing reports of substantially higher potentials of up to +70 mV in the cupula of ampullae of the semicircular canals. Similarly, in the auditory system, a high (K+) is observed. However, in contrast to the vestibular system, the positive endolymphatic potential varies more substantially between vertebrates, ranging from near zero mV to approximately +100 mV. The tissues generating endolymph in the inner ear show considerable differences in cell types and location. So-called dark cells and the possibly homologous ionocytes in fish appear to be the common elements, but there is always at least one additional cell type present. To inspire research in this field, we propose a classification for these cell types and discuss potential evolutionary relationships. Their molecular repertoire is largely unknown and provides further fertile ground for future investigation. Finally, we propose that the ultimate selective pressure for an increased endolymphatic potential, as observed in mammals and to a lesser extent in birds, is specifically to maintain the AC component of the hair-cell receptor potential at high frequencies. In summary, we identify intriguing questions for future directions of research into the molecular and cellular basis of the endolymph in the different compartments of the inner ear. The answers will provide important insights into evolutionary and developmental processes in a sensory organ essential to many species, including humans.

Differences in molecular mechanisms of K+ clearance in the auditory sensory epithelium of birds and mammals.

Mechanoelectrical transduction in the vertebrate inner ear is a highly conserved mechanism that is dependent on K+ influx into hair cells. Here, we investigated the molecular underpinnings of subsequent K+ recycling in the chicken basilar papilla and compared them with those in the mammalian auditory sensory epithelium. As in mammals, the avian auditory hair cell uses KCNQ4, KCNMA1 and KCNMB1 in its K+ efflux system. Expression of KCNQ1 and KCNE1 suggests an additional efflux apparatus in avian hair cells. Marked differences were observed for K+ clearance. In mammals, KCC3, KCC4, Kir4.1 and CLC-K are present in supporting cells. Of these, only CLC-K is expressed in avian supporting cells. Instead, they possess NKCC1 to move K+ across the membrane. This expression pattern suggests an avian clearance mechanism reminiscent of the well-established K+ uptake apparatus present in inner ear secretory cells. Altogether, tetrapod hair cells show similar mechanisms and supporting cells show distinct molecular underpinnings of K+ recycling.

Molecular bases of K+ secretory cells in the inner ear: shared and distinct features between birds and mammals.

In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K+] is always present in the inner ear endolymph. Here, we show that Kir4.1, which is required in the mammalian stria vascularis to generate the highly positive EP, is absent in the functionally equivalent avian tegmentum vasculosum. In contrast, the molecular repertoire required for K+ secretion, specifically NKCC1, KCNQ1, KCNE1, BSND and CLC-K, is shared between the tegmentum vasculosum, the vestibular dark cells and the marginal cells of the stria vascularis. We further show that in barn owls, the tegmentum vasculosum is enlarged and a higher EP (~+34 mV) maintained, compared to other birds. Our data suggest that both the tegmentum vasculosum and the stratified stria vascularis evolved from an ancestral vestibular epithelium that already featured the major cell types of the auditory epithelia. Genetic recruitment of Kir4.1 specifically to strial melanocytes was then a crucial step in mammalian evolution enabling an increase in the cochlear EP. An increased EP may be related to high-frequency hearing, as this is a hallmark of barn owls among birds and mammals among amniotes.

Functional Role of γ-Crystallin N in the Auditory Hindbrain.

γ-crystallins are major components of the vertebrate lens but show expression in other tissues as well. Their extralenticular functions remain so far unclear. Here, we explored such roles in the rodent superior olivary complex in which previous analysis demonstrated developmentally regulated expression of Crygd, Cryge and Crygn. Immunohistochemistry with novel antibodies against Crygd/e and Crygn indicate that expression of Crygd/e was moderate and varied between the perinatal superior olivary complex of mice, rats, and gerbils. Crygn-immunoreactivity was more robust and consistently highest in the medial nucleus of the trapezoid body, but also present in other nuclei of the superior olivary complex. To analyze the function of Crygn in the auditory hindbrain, we used a Crygn allele with a floxed exon 2. Upon pairing with Egr2::Cre mice, exon 2, encoding the first two greek key motifs of Crygn, was deleted in the developing auditory hindbrain. Anatomical analysis of these mice revealed a 20% volume reduction in the medial nucleus of the trapezoid body and a 7% reduction in the lateral superior olive at postnatal day 25. This was due to cell loss between postnatal days 4 and 25, whereas cell size was unaffected. Auditory brainstem responses showed normal threshold but a significant increase in the amplitude of wave IV. Crygn is hence required for postmigratory survival and proper function of auditory hindbrain neurons. These results ascertain for the first time an essential extralenticular role for γ-crystallins in vivo.