Sox2-CreER mice are useful for fate mapping of mature, but not neonatal, cochlear supporting cells in hair cell regeneration studies

Abstract

Studies of hair cell regeneration in the postnatal cochlea rely on fate mapping of supporting cells. Here we characterized a Sox2-CreER knock-in mouse line with two independent reporter mouse strains at neonatal and mature ages. Regardless of induction age, reporter expression was robust, with CreER activity being readily detectable in >85% of supporting cells within the organ of Corti. When induced at postnatal day (P) 28, Sox2-CreER activity was exclusive to supporting cells demonstrating its utility for fate mapping studies beyond this age. However, when induced at P1, Sox2-CreER activity was also detected in >50% of cochlear hair cells, suggesting that Sox2-CreER may not be useful to fate map a supporting cell origin of regenerated hair cells if induced at neonatal ages. Given that this model is currently in use by several investigators for fate mapping purposes, and may be adopted by others in the future, our finding that current protocols are effective for restricting CreER activity to supporting cells at mature but not neonatal ages is both significant and timely.

Figure 1

A,B. Sox2-CreER labels >85% supporting cells and >50% inner and outer hair cells in the organ of Corti when induced at P0-1. C,D. Sox2CreER labels only supporting cells in the organ of Corti when induced at P28. Wholemount cochlear confocal images (A,C) and quantification (B,D) of Sox2-CreER; ROSA-CAG-Stop-floxed-tdTomato (Ai14) when induced at P0-P1 and analyzed at P8, or induced at P28 and analyzed at P35. Hair cells are labeled with Myo7a antibody (white) in the P0-P1 induced samples (A), and in the P28 induced samples (C). Sox2 immunostaining (green) labels supporting cells, and tdTomato (red) labels Cre activity. (B and D). Percentages of tdTomato+ cells in each subtype of supporting cells (Inner phallengeal cells [Iph], Inner pillar cells [IPC], outer pillar cells [OPC], Deiters’ cells [DC], Hensen cells [Hens]) and hair cells (HCs). Scale bars = 20 μm. Error bars = ±1 standard error in three independent mice.

Figure 2. Sox2-CreER labels both supporting cells and hair cells when induced at P1.

Tamoxifen was intraperitoneally injected into Sox2-CreER; mTmG mice at P1 and cochleae were analyzed at P38. A. The whole mount of apical cochlear turn with mGFP (green) and mtdTomato (red) fluorescence and Myo7a immunostaining (blue) using LSM700 confocal laser scanning microscope. B. The optical xz-plane of the line in A. Arrowheads label mGFP⁺ hair bundles. Scale = 20 μm. C. Percentages of mGFP⁺ hair cells in a 300-μm region from the apical turn. Error bars = ±1 standard error, N = 3 independent mice.