Quantitative Analysis of Supporting Cell Subtype Labeling Among CreER Lines in the Neonatal Mouse Cochlea

Four CreER lines that are commonly used in the auditory field to label cochlear supporting cells (SCs) are expressed in multiple SC subtypes, with some lines also showing reporter expression in hair cells (HCs). We hypothesized that altering the tamoxifen dose would modify CreER expression and target subsets of SCs. We also used two different reporter lines, ROSA26 ^tdTomato and CAG-eGFP, to achieve the same goal. Our results confirm previous reports that Sox2 ^CreERT2 and Fgfr3-iCreER ^T2 are not only expressed in neonatal SCs but also in HCs. Decreasing the tamoxifen dose did not reduce HC expression for Sox2 ^CreERT2 , but changing to the CAG-eGFP reporter decreased reporter-positive HCs sevenfold. However, there was also a significant decrease in the number of reporter-positive SCs. In contrast, there was a large reduction in reporter-positive HCs in Fgfr3-iCreER ^T2 mice with the lowest tamoxifen dose tested yet only limited reduction in SC labeling. The targeting of reporter expression to inner phalangeal and border cells was increased when Plp-CreER ^T2 was paired with the CAG-eGFP reporter; however, the total number of labeled cells decreased. Changes to the tamoxifen dose or reporter line with Prox1 ^CreERT2 caused minimal changes. Our data demonstrate that modifications to the tamoxifen dose or the use of different reporter lines may be successful in narrowing the numbers and/or types of cells labeled, but each CreER line responded differently. When the ROSA26 ^tdTomato reporter was combined with any of the four CreER lines, there was no difference in the number of tdTomato-positive cells after one or two injections of tamoxifen given at birth. Thus, tamoxifen-mediated toxicity could be reduced by only giving one injection. While the CAG-eGFP reporter consistently labeled fewer cells, both reporter lines are valuable depending on the goal of the study.