Abstract
Lgr5+ cochlear supporting cells (SCs) have been reported to be hair cell (HC) progenitor cells that have the ability to regenerate HCs in the neonatal mouse cochlea, and these cells are regulated by Wnt signaling. Frizzled-9 (Fzd9), one of the Wnt receptors, has been reported to be used to mark neuronal stem cells in the brain together with other markers and mesenchymal stem cells from human placenta and bone marrow. Here we used Fzd9-CreER mice to lineage label and trace Fzd9+ cells in the postnatal cochlea in order to investigate the progenitor characteristic of Fzd9+ cells. Lineage labeling showed that inner phalangeal cells (IPhCs), inner border cells (IBCs), and third-row Deiters’ cells (DCs) were Fzd9+ cells, but not inner pillar cells (IPCs) or greater epithelial ridge (GER) cells at postnatal day (P)3, which suggests that Fzd9+ cells are a much smaller cell population than Lgr5+ progenitors. The expression of Fzd9 progressively decreased and was too low to allow lineage tracing after P14. Lineage tracing for 6 days in vivo showed that Fzd9+ cells could also generate similar numbers of new HCs compared to Lgr5+ progenitors. A sphere-forming assay showed that Fzd9+ cells could form spheres after sorting by flow cytometry, and when we compared the isolated Fzd9+ cells and Lgr5+ progenitors there were no significant differences in sphere number or sphere diameter. In a differentiation assay, the same number of Fzd9+ cells could produce similar amounts of Myo7a+ cells compared to Lgr5+ progenitors after 10 days of differentiation. All these data suggest that the Fzd9+ cells have a similar capacity for proliferation, differentiation, and HC generation as Lgr5+ progenitors and that Fzd9 can be used as a more restricted marker of HC progenitors.
Highlights
Sensorineural hearing loss is mainly caused by hair cell (HC) loss and is one of the most common health problems around the world
Tamoxifen was I.P. injected into P3 Fzd9-CreER/Rosa26-tdTomato double-positive mice to activate cre, and Fzd9+ cells were labeled by tdTomato fluorescence after 48 h (Figure 1A). tdTomato-labeled Fzd9+ cells were observed from the apex to the base in the cochlea (Figure 1B), and tdTomato-labeled Fzd9+ cells were found in the supporting cells (SCs) layer (Figures 1C,D), including the inner phalangeal cells (IPhCs) and inner border cells (IBCs) and to a lesser extent the third-row Deiters’ cells (DCs) as shown by the high-resolution images (Figures 1E,F, Supplementary Figure S2)
The cell types and number of Fzd9+ cells were much lower than Lgr5+ progenitors, which suggests that Fzd9+ cells might be a subset of HC progenitors
Summary
Sensorineural hearing loss is mainly caused by hair cell (HC) loss and is one of the most common health problems around the world. Recent studies have shown that HCs can be regenerated in newborn mice (Chai et al, 2012; Shi et al, 2012; Bramhall et al, 2014; Wang et al, 2015; Li et al, 2016; You et al, 2018; Zhang et al, 2018) This regenerative ability is quickly lost as the mice age (BerminghamMcDonogh and Reh, 2011; Chai et al, 2012; Shi et al, 2012; Bramhall et al, 2014; Cox et al, 2014). Lgr is expressed in third-row Deiters’ cells (DCs), inner pillar cells (IPCs), inner phalangeal cells (IPhCs), and part of the lateral greater epithelial ridge (GER) cells, which is a large cell population and contains many different cell types (Chai et al, 2011, 2012; Shi et al, 2012)
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