Mammals have adapted to live in the darkest of caves and the deepest oceans, and from the highest mountains to the plains. Along the way, mammals have also adapted a remarkable capacity in their sense of hearing, from the high-frequency echolocation calls of bats to low frequency whale songs. Even our best friend companion animals, dogs, have developed a hearing range twice as wide as people.

Assuming that these adaptations have a root genetic cause, a team of scientists led by Lucia Franchini of the National Council of Scientific and Technological Research (CONICET), in Buenos Aires, Argentina, have made it their goal to identify the genetic bases underlying the evolution of the inner ear in mammals. Their latest findings underscored the promise of their approach, which identified two new genes involved in hearing. The study was published in the advanced online edition of Molecular Biology and Evolution.

"This paper builds on the premise that the evolution of mammalian inner ear hearing related novelties should leave a discoverable trace of adaptive molecular signature," said Franchini. "This work highlights the usefulness of evolutionary studies to pinpoint novel key functional genes."

The basic processes of hearing in different mammalian species is the same. The auditory system of mammals is characterized by a middle ear composed of three ossicles (the incus (anvil), malleus (hammer) and stapes (stirrup), which funnels sound to the inner ear.

Franchini's group focused on the inner ear, which turns changes in sound intensity into electrical signals that the brain can process. Within the inner ear is the snail-shaped cochlea that transforms sound waves into nerve impulses, including an auditory organ of Corti that possesses two types of specialized sensory hair cells (HCs), inner (IHCs) and outer hair cells (OHCs).

"In the mammalian cochlea, IHCs and OHCs display a clear division of labor," explains Franchini. "The IHCs receive and relay sound information behaving as the true sensory cells, while OHCs amplify sound information. Thus, IHCs which are the primary transducers, release glutamate to excite the sensory fibers of the cochlear nerve and OHCs act as biological motors to amplify the motion of the sensory epithelium."

In their study, they used a two-pronged approach, complementing in silico gene comparisons with follow-up experimental studies, to gain a more complete understanding of the genetic circuitry behind mammalian inner ear adaptations.

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"These functional and morphological innovations in the mammalian inner ear contribute to its unique hearing capacities," said lead author Lucia Franchini. However, the genetic bases underlying the evolution of this mammalian landmark are poorly understood. We propose that the emergence of morphological and functional innovations in the mammalian inner ear could have been driven by adaptive molecular evolution."

First, they took advantage of extensive gene expression databases to perform software-based, or, in silico comparative studies of 1,300 genes to identify genes that may have been positively selected to help mammals adapt over evolutionary time. In total, they found 13%, or 165 inner ear genes that may have been selected for adaptation.

"This analysis indicated that both IHCs and OHCs went through similar levels of gene adaptive evolution probably underlying the morphological and functional remodelling that both cellular types underwent in the mammalian lineage," said Franchini.

"Notably we found that analysing functional categories of positively selected genes the most enriched functional term were 'cytoskeletal protein binding' and 'structural constituent of the cytoskeleton'. These findings indicate that the OHC genes that underwent positive selection could have contributed to the acquisition of the highly specialized cytoskeleton present in these cells that underlies its distinctive functional properties, including somatic electromotility."

Next, they experimentally tested hearing gene functions in a series of mouse studies. Among these, they focused on two previously unknown inner ear genes: STRIP2 (from Striatin Interacting Protein 2) and ABLIM2 (Actin Binding LIM domain 2), which were functionally characterized by generating novel strains of mutant mice by using CRISPR/Cas9 technology. In each case, they used CRISPR to turn off part of the normal gene function to see how it affected the hearing genetic circuitry.

"We performed auditory functional studies of Strip2 and Ablim2 newly generated mutant mice by means of two complementary techniques that allow differential diagnosis of OHC versus IHC/neuronal dysfunction throughout the cochlea," said Franchini. "To evaluate the integrity of the hearing system we recorded ABRs (Auditory Brainstem Responses) that are sound-evoked potentials generated by neuronal circuits in the ascending auditory pathways. We also evaluated the OHCs function through distortion product otoacoustic emissions (DPOAE) testing."

They discovered that Strip2 likely plays a functional role in the first synapse between IHCs and nerve fibers. Moreover, when they at the cochlear sensory epithelium, they found a significant reduction in auditory-nerve synapses. In contrast, the mutant studies of Ablim2 suggest that the absence of Ablim2 does not affect either cochlear amplification or auditory nerve function.

"In summary, through this evolutionary approach we discovered that STRIP2 underwent strong positive selection in the mammalian lineage and plays an important role in the physiology of the inner ear," said Franchini. "Moreover, our combined evolutionary and functional studies allow us to speculate that the extensive evolutionary remodeling that this gene underwent in the mammalian lineage provided an adaptive value. Thus, our study is a proof of concept that evolutionary approaches paired with functional studies could be a useful tool to uncover new key players in the function of organs and tissues."