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. 2015 Jul 1;523(10):1474-87.
doi: 10.1002/cne.23747. Epub 2015 Mar 10.

Characterization of a unique cell population marked by transgene expression in the adult cochlea of nestin-CreER(T2)/tdTomato-reporter mice

Cynthia L Chow  1   2 , Weixiang Guo  2 , Parul Trivedi  2 , Xinyu Zhao  2   3 , Samuel P Gubbels  2   4
Affiliations

Characterization of a unique cell population marked by transgene expression in the adult cochlea of nestin-CreER(T2)/tdTomato-reporter mice

Cynthia L Chow et al. J Comp Neurol. .

Abstract

Hair cells in the adult mammalian cochlea cannot spontaneously regenerate after damage, resulting in the permanency of hearing loss. Stem cells have been found to be present in the cochlea of young rodents; however, there has been little evidence for their existence into adulthood. We used nestin-CreER(T2)/tdTomato-reporter mice to trace the lineage of putative nestin-expressing cells and their progeny in the cochleae of adult mice. Nestin, an intermediate filament found in neural progenitor cells during early development and adulthood, is regarded as a multipotent and neural stem cell marker. Other investigators have reported its presence in postnatal and young adult rodents; however, there are discrepancies among these reports. Using lineage tracing, we documented a robust population of tdTomato-expressing cells and evaluated these cells at a series of adult time points. Upon activation of the nestin promoter, tdTomato was observed just below and medial to the inner hair cell layer. All cells colocalized with the stem cell and cochlear-supporting-cell marker Sox2 as well as the supporting cell and Schwann cell marker Sox10; however, they did not colocalize with the Schwann cell marker Krox20, spiral ganglion marker NF200, nor glial fibrillary acidic acid (GFAP)-expressing supporting cell marker. The cellular identity of this unique population of tdTomato-expressing cells in the adult cochlea of nestin-CreER(T2)/tdTomato mice remains unclear; however, these cells may represent a type of supporting cell on the neural aspect of the inner hair cell layer.

Keywords: AB_10013382; AB_10015251; AB_10064079; AB_149792; AB_2195374; AB_2251134; AB_2286684; AB_2314882; AB_306298; AB_396354; inner ear; mouse; regeneration; stem cell; supporting cell.

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Conflict of interest statement

Conflict of Interest

The authors declare no potential conflict of interest.

Figures

Figure 1
Figure 1
Generation of transgenic mice and activation of tdTomato following tamoxifen (TMX) administration. A: Nestin-CreERT2 mice and Rosa-tdTomato (Ai14) reporter mice were bred to create transgenic offspring. B: CreERT2 is expressed under the control of the nestin promoter. The expression of nestin-CreERT2 is inducible by means of tamoxifen (TMX) injection, which activates the estrogen receptor fused to Cre (CreERT2), allowing it to enter the nucleus, recombine the LoxP sites to remove a stop codon, and express tdTomato in tissues expressing Cre-recombinase. C: Timeline of experiment. Mice were given 5 injections of tamoxifen (TMX) on 5 subsequent days, after which their tissue was harvested 1, 7, 14, 35 or 56 days following the last TMX injection.
Figure 2
Figure 2
Cochlea from an adult nestin-CreERT2/tdTomato-reporter mouse 56 days post tamoxifen injection. Low power macroscopic imaging analysis showed a pattern of tdTomato expression on the neural aspect of Sox2-positive supporting cells. An apical-basal gradient was not observed in tdTomato-positive cells. Image was acquired on a Lightsheet Z.1 by Carl Zeiss Microscopy GmbH with a 5x/0.16 EC Plan-NEOFLUAR objective. Scale bar: 500μm.
Figure 3
Figure 3
TdTomato-positive cells are found throughout the adult mammalian inner ear for all time points examined. A: Orientation of the organ of Corti. B–D: TdTomato-positive cells are observed throughout the cochlea in the apex (B), mid (C), and base (D) 56 days after the last tamoxifen injection. Images were acquired using a Nikon A1R-A1 confocal microscope. E-AH: High-power images showing tdTomato-positive cells in the normal mammalian inner ear 1 day, 7 days, 14 days, 35 days, and 56 days after the last tamoxifen injection (G, L, Q, V, AA respectively). The morphology of the tdTomato-positive cells appeared consistent, having oval cells bodies with extensions protruding upward towards the inner hair cell layer; however, some tdTomato-positive cells appeared to have slightly different morphologies with elongated oval bodies and/or widening protrusions near the apical end. Cochlear hair cells, labeled with Myosin VIIa, reveal a normal configuration with three rows of outer hair cells and one row of inner hair cells (H, M, R, W, AB, AG). TdTomato co-localizes with the stem cell and supporting cell marker Sox2 (I, N, S, X, AC). An adult control nestin-CreERT2/tdTomato-reporter mouse confirmed no tdTomato was expressed in the cochlea in the absence of tamoxifen (AF). Images were acquired using a Nikon A1R-A1 confocal microscope. Scale bar: 500 μm in B–D; 50 μm in E-AH.
Figure 4
Figure 4
Cellular organization of tdTomato-positive cells in the normal mammalian inner ear. A: Orientation of the organ of Corti. B–C: Cross sectional view of a cochlea 35 days after the last tamoxifen injection reveals one row of inner hair cells and three rows of outer hair cells along with a normal compliment of cells labeled with the stem cell and supporting cell marker Sox2. D: Apical region of the cochlea 7 days after the last tamoxifen injection. TdTomato-positive cells are located below and medial to the inner hair cell layer and co-localize with the stem cell and supporting cell marker Sox2. Scale bar: 10 μm in B–D.
Figure 5
Figure 5
Characterization of tdTomato-positive cells. A: In adult murine cochlear tissue, cells expressing Krox20 co-expressed with the Schwann cell and supporting cell marker Sox10 (arrows indicate double-layered cells with overlap shown in white). A smaller percentage of Sox10-positive cells expressed Krox20, suggesting Krox20 is a more precise Schwann cell marker B: NF200 antibody recognized a single band of about 200 kDa using adult murine brain lysates and cochlear tissues. C–G: 1 day after the last tamoxifen injection. The supporting cell and Schwann cell marker Sox10 co-localized with all tdTomato-positive cells. H–L: 14 days after the last tamoxifen injection. Krox20, a Schwann cell marker, did not co-localize with tdTomato-positive cells. M–Q: 14 days after the last tamoxifen injection. The spiral ganglion cell marker NF200 did not co-localize with tdTomato-positive cells. R–V: 7 days after the last tamoxifen injection. GFAP (expressed in border and inner phalangeal cell sub-populations of supporting cells) did not co-localize with tdTomato-positive cells. Images were acquired using a Nikon A1R-A1 confocal microscope. Scale bar: 10 μm in A; 50 μm in panels F, K, P, U; and 10μm in panels G, L, Q, V.
Figure 6
Figure 6
Statistical analysis. A: Box plot displaying the averages and spread of data (number of cells per high powered field) for each time point (Day). The mean number of tdTomato-positive cells increased from day 1 after the last tamoxifen injection to day 35, after which there was a slight decrease at day 56. B: Post-hoc tests using Tukey HSD comparing each time point (Day) to a comparison time point (Comparison Day) and the significance. There was no significant difference for the number of tdTomato-positive cells between days 1 and 7 post tamoxifen injection, and no significant difference between days 7 and 56. The only statistically significant difference seen was between day 1 and days 14 to 56. For all analyses, differences were considered significant if P < 0.05.
Figure 7
Figure 7
The cellular organization of the organ of Corti consists of several supporting cells including: Deiters’, Hensen’s, pillar, border, and inner phalangeal cells, in addition to inner and outer mechanosensory hair cells. TdTomato-positive cells appeared under and medial to the inner hair cell layer in the neural aspect.
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