Expression of LHX3 and SOX2 during mouse inner ear development

Abstract

A cascade of transcription factors is believed to regulate the coordinate differentiation of primordial inner ear cells into the subtypes of hair cells and supporting cells. While candidate genes involved in this process have been identified, the temporal and spatial patterns of expression of many of these have not been carefully described during the extended period of inner ear development and functional maturation. We systematically examined the expression of two such transcription factors, LHX3 and SOX2, from the time of hair cell terminal mitoses into adulthood. We show that LHX3 is expressed specifically in auditory and vestibular hair cells soon after terminal mitoses and persists into the adult in vestibular hair cells. While SOX2 expression is widespread in the inner ear sensory epithelia prior to hair cell differentiation, it has a unique pattern of expression in the mature auditory and vestibular organs.

Figure 1

Embryonic day 13-16. Immunofluorescence was used to localize the expression of SOX2 (green, A, E, I, M), ATOH1 (green, B, F, J, N, Q), LHX3 (green, C, G, K, O, R) and MYO6 (green, D, H, L, P, S) in the vestibular and auditory sensory epithelia of the embryonic mouse. E13 (A-D), E14 (E-H), E15 (I-P) E16 (Q-S). In each image, nuclei are counterstained with bisbenzimide (pseudocolored red A-B and I-S; blue E-H). Expression of each protein was detected in all five vestibular epithelial patches at these ages. In panels E-H, timed-mated pregnant animals were injected with BrdU at E13.5 and sacrificed 8 hours later. Adjacent sections were double-labeled with antibodies for BrdU (red) and the indicated marker (green). For E-H, each image shows the brightest point projection of a z-series confocal stack. Double-labeled cells are seen only in the SOX2 panel (E). A mid-modiolar section of the E15 basal cochlea is shown (M-P), however, expression of each gene is comparable in the later maturing, more apical regions (i.e., only SOX2 is detected, not shown). One day later, ATOH1 (Q), LHX3 (R) and MYO6 (S) are expressed in inner hair cells of the basal cochlea. ATOH1 expression is also faintly detectable in OHC at this age (closed arrowheads Q). HC indicates the depth of the hair cell layer; SC indicates the depth of the support cell layer. GER = greater epithelial ridge, LER = lesser epithelial ridge, OHC = outer hair cell, IHC = inner hair cell. The dashed lines in E-H indicate the borders of the sensory epithelium. The asterisks indicate non-specific label (Q, S). Scale bars shown in D, H, P and S = 20 μm in A-D, E-H, I-P and Q-S, respectively.

Figure 2

Embryonic day 17. Immunofluorescence was used to localize the expression of SOX2 (green, A, E, I, M; red, Q, R), ATOH1 (green, B, F, J, N), LHX3 (green, C, G, K, O), MYO6 (green, D, H, L, P, Q) and PROX1 (green, R) in the vestibular and auditory sensory epithelia of the embryonic mouse at E17. In each image, nuclei are counterstained with bisbenzimide (pseudocolored red A-P; blue Q,R). Expression of each protein was detected in all five vestibular epithelial patches although only a representative series of sequential sections of the saccule is shown (A-D). Mid-modiolar sections through the base, middle and apex of the cochlea are shown (E-R). Note that the inner phalangeal cells and more medial support cells express SOX2, but not PROX1 (Q, R). Downward and upward arrows indicate hair cells (IHC= Inner hair cell, OHC= Outer hair cell) and support cell (SC, IPhC = Inner phalangeal cell, PC = Pillar cells) nuclei, respectively. HC indicates the depth of the hair cell layer. GER = greater epithelial ridge, LER = lesser epithelial ridge. BM indicates the level of the basement membrane, below the support cell nuclei. Scale bars in P and R = 20 μm and 10 μm respectitvely for panels A-P and Q, R. Asterisks in O indicate non-specific overlying debris.

Figure 3

Postnatal day 2. Immunofluorescence was used to localize the expression of SOX2 (green, A, E, I, M), ATOH1 (green, B, F, J, N), LHX3 (green, C, G, K, O) and MYO6 (green, D, H, L, P) in the vestibular and auditory sensory epithelia of the embryonic mouse at postnatal day 2. For each image, nuclei were counterstained with bisbenzimide and pseudo-colored red. Expression of each protein was detected in all five vestibular epithelial patches although only a representative section of the saccule is shown (A-D). Representative mid-modiolar sections through the base, middle and apex of the cochlea are shown. HC indicates the depth of the hair cell layer; SC indicates the depth of the support cell layer (A, D). Arrowheads in A indicate SOX2 expressing nuclei in the HC layer. Downward and upward arrows in G and I indicate hair cell (IHC= Inner hair cell, OHC= Outer hair cell) and support cell (SC) nuclei, respectively. Scale bar (P) = 20 μm in all panels.

Figure 4

Adult. Immunofluorescence and immunohistochemistry were used to localize the expression of SOX2, MYO7A (A, D, G), LHX3 (B, E, H) and MYO6 (C, F, I) in the vestibular and auditory sensory epithelia of adult mice. A representative mid-modiolar section through the middle turn of cochlea is shown for each antibody (G, H, I). LHX3 and MYO6 while were analyzed by immunofluorescence (A, D, G; red = SOX2, green = MYO7A, blue = bisbenzimide). Downward arrowheads in A, D and G indicate SOX2-expressing nuclei in the HC layer. Upward pointing arrows in G and I indicate subtypes of support cells (SC). PC= pillar cells; DC= Deiters' cells; IPhC= Inner phalangeal cells. IHC= Inner hair cells; OHC= Outer hair cells;. Bars in A-C indicate the depth of the hair cell (HC) and support cell (SC) layers. Scale bar (I) = 20 μm in all panels.

Figure 5

Confocal images of SOX2 expression in the adult inner ear. Whole-mount preparations of normal adult mouse saccule (A, B) or cochlea (C, D) were immunolabeled for SOX2 (red, A-D) and the calcium-binding protein calbindin (green, A) which labels afferent calyces and delineates the striolar region, or the HC-specific marker calbindin (green, C, D),. Nuclei were counterstained with bisbenzimide (blue). Each image shows the brightest point projection of a z-series stack taken parallel to the lumenal surface of the epithelium (step size = 1 μm). For images of the saccule, adjacent segments of a single stack spanning through either the lumenal hair cell (A) or more basal support cell nuclear layers (B) are shown. In panel A, Type I HCs in the striolar region are surrounded by calbindin-positive nerve fibers and are SOX2-negative. Hair cells in the extrastriolar region are either SOX2-positive or SOX2-negative. In B, all support cell nuclei in the basal layer are SOX2 positive. Calbindin labeling of afferent fibers is not shown in B for clarity. In the organ of Corti images (C, D), the projection spans the full thickness of the epithelium to emphasize the medial and lateral extent of SOX2 expression relative to the position of hair cells. Arrowheads indicate the 3 rows of outer hair cells (OHC) and the single row of inner hair cells (IHC). The red (SOX2) and green (Parvalbumin) channels of panel 5C are separated in the left and right halves of panel 5D. TC/LTC, Tectal cell/Lateral tunnel cell. Scale bar = 20 μm for A; 10 μm for B-C.