Characterization of the Transcriptomes of Lgr5+ Hair Cell Progenitors and Lgr5- Supporting Cells in the Mouse Cochlea

Complete knowledge of all direct and indirect interactions between proteins in a given cell would represent an important milestone towards a comprehensive description of cellular mechanisms and functions. Although this goal is still elusive, considerable progress has been made—particularly for certain model organisms and functional systems. Currently, protein interactions and associations are annotated at various levels of detail in online resources, ranging from raw data repositories to highly formalized pathway databases. For many applications, a global view of all the available interaction data is desirable, including lower-quality data and/or computational predictions. The STRING database (http://string-db.org/) aims to provide such a global perspective for as many organisms as feasible. Known and predicted associations are scored and integrated, resulting in comprehensive protein networks covering >1100 organisms. Here, we describe the update to version 9.1 of STRING, introducing several improvements: (i) we extend the automated mining of scientific texts for interaction information, to now also include full-text articles; (ii) we entirely re-designed the algorithm for transferring interactions from one model organism to the other; and (iii) we provide users with statistical information on any functional enrichment observed in their networks.

Loss of cochlear hair cells in mammals is currently believed to be permanent, resulting in hearing impairment that affects more than 10% of the population. Here, we developed two genetic strategies to ablate neonatal mouse cochlear hair cells in vivo. Both Pou4f3(DTR/+) and Atoh1-CreER™; ROSA26(DTA/+) alleles allowed selective and inducible hair cell ablation. After hair cell loss was induced at birth, we observed spontaneous regeneration of hair cells. Fate-mapping experiments demonstrated that neighboring supporting cells acquired a hair cell fate, which increased in a basal to apical gradient, averaging over 120 regenerated hair cells per cochlea. The normally mitotically quiescent supporting cells proliferated after hair cell ablation. Concurrent fate mapping and labeling with mitotic tracers showed that regenerated hair cells were derived by both mitotic regeneration and direct transdifferentiation. Over time, regenerated hair cells followed a similar pattern of maturation to normal hair cell development, including the expression of prestin, a terminal differentiation marker of outer hair cells, although many new hair cells eventually died. Hair cell regeneration did not occur when ablation was induced at one week of age. Our findings demonstrate that the neonatal mouse cochlea is capable of spontaneous hair cell regeneration after damage in vivo. Thus, future studies on the neonatal cochlea might shed light on the competence of supporting cells to regenerate hair cells and on the factors that promote the survival of newly regenerated hair cells.

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.