Article Figures & Data
Figures
- Figure 1.
S1P receptors are expressed in the cochlea. A, RT-PCR analysis demonstrates that three high-affinity S1P receptors (S1P1–3) are expressed in the murine cochlea from neonates into adulthood. β-Actin primers served as a cDNA loading control. The lung cDNA served as a positive control for the primers, because all three genes are known to be expressed there (Ishii et al., 2001). All cDNA samples (+) were run in parallel with a negative control template prepared without the addition of reverse transcriptase (–). B, qRT-PCR reveals the developmental dynamics of S1P receptor expression in the cochlea. Expression level is represented as copies of receptor mRNA per copy of β-actin mRNA. Values are averages of three individual mice at each time point. Error bars indicate SD. C, In situ hybridization of this P19 cochlea indicates that S1P2 is expressed in the organ of Corti in the supporting cells (arrowheads), primarily those underlying the hair cells. There is little detectable signal in the hair cells (arrows), but it is observed occasionally as shown here (white arrow). D, Sense-strand control demonstrating the specificity of the S1P2 probe. E, In situ hybridization of this P4 cochlea with an S1P3-specific antisense probe shows that S1P3 is expressed throughout the cochlea and spiral ganglia. Its pattern suggests expression in the vasculature and possibly the cochlear glia. F, Higher-magnification view emphasizing that little expression of S1P3 is observed inside the organ of Corti (arrow). Insets show sense-strand control probes in E and F.
- Figure 2.
Loss of S1P2 results in vestibular and cochlear defects. A, s1p2/s1p3 double-null mice display a distinctive head tilt posture characteristic of vestibular lesions. The age of onset varies but is usually detectable before 6 months of age. B, When suspended by the tail, wild-type mice will stretch their forelimbs downward toward the ground. Mice with vestibular defects curl upward, often landing on their heads when returned to the ground. s1p2/s1p3 double-null mice display this behavior, with an age-related increase in penetrance starting at 2 months and occurring uniformly by 6 months of age. C, Wild-type and s1p3−/− mice invariably swim for the full 1 min test period when placed in a beaker of water. s1p2/s1p3 double-null mice fail this test at an increasing frequency with age, with a near-complete penetrance by 18 weeks. D, Mice with vestibular defects have difficulty maintaining balance on their hindlimbs and, rather than engaging in normal exploratory behavior by rearing, instead arch their backs to elevate their noses. The marked decrease in rearing frequency noted in these 12- to 14-week-old s1p2/s1p3 double-null mice is consistent with vestibular dysfunction (**p < 0.01). E, S1P2 null mice completely lack a detectable response to an acoustic startle stimulus. Heterozygous and s1p3 null mice respond normally with startle reflex thresholds of 90–100 decibels. s1p2 null mice uniformly fail to respond to even the strongest stimulus from the youngest age tested (4 weeks). Response shown is measured in arbitrary force units. Error bars indicate SD.
- Figure 3.
Hair cell development is normal in s1p2/s1p3 double-null mice. A, B, Vestibular maculas of wild-type and s1p2−/−/s1p3−/− mice at P1. The sensory epithelium of the knock-out is normal in appearance with normal cell polarity, innervation, and stereociliary projections. C, D, Cochleas of wild-type and s1p2−/−/s1p3−/− mice at P1. All structures of the neonatal cochlea appear to develop normally in the knock-out mouse. Note the presence of one inner and three outer hair cells (arrows) in the developing organ of Corti. Scale bar: (in A) A–D, 50 μm. E–J, Surface preparation of wild-type and s1p2−/−/s1p3−/− organs of Corti. Tissue was obtained at P4 from the basal turn of the cochlea, stained with phalloidin to label F-actin in the stereocilia (E, F), and stained for CDH23, a marker for differentiating hair cells (G, H). The bottom panels (I, J) show a merge of the two images. Stereociliary bundles appear to be normal in size and morphology in the knock-out animal. Scale bar: (in E) E–J, 5 μm.
- Figure 4.
Loss of S1P2/S1P3 causes progressive degeneration of vestibular end organs and cochleas. A, B, Vestibular maculas of an 8-month-old s1p2−/−/s1p3−/− mouse and heterozygous littermate. The knock-out epithelial layer is markedly disorganized and lacks afferent calyces (arrows) and stereocilia (arrowheads). C, This 2-month-old heterozygous mouse displays normal morphology throughout the length of the organ of Corti. Note the presence of the one inner and three outer hair cells (arrows). D, This s1p2−/−/s1p3−/− littermate bears the characteristic degeneration of the organ of Corti, most strikingly in the basal turn as shown here. Note the absence of all cells of the organ of Corti, including the hair cells (*). F, This image of a 4-month-old cochlea illustrates the characteristic basal-to-apical degeneration seen in our s1p2−/−/s1p3−/− mice. Note that cells are lost completely in the basal turn (*) (as seen in D) and are present but disorganized in the apical turns (arrowhead) as compared with similar structures in the heterozygous littermate (E). Also note the absence of afferent neurons in the spiral ganglia (arrows) that is coincident with loss of the organ of Corti.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental legends
- supplemental material - Figure S1
- supplemental material - Movie S2
- supplemental material - Movie S3
