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First published online 1 September 2005
doi: 10.1242/dev.02002

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The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear

¹ The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 USA
² Institut für Molekularbiologie, Medizinische Hochschule, 30625 Hannover, Germany
³ Departments of Molecular Biology and Pharmacology and Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (87K): [in a new window]   Fig. 1. Graded increase in hair cell formation with a reduction in Notch ligand gene dosage. (A-D) Lectin-stained cochleae with only single null or hypomorphic mutations in the Dll1 or Jag2 genes show relatively normal hair cell numbers and patterning, although occasional extra inner or outer hair cells are observed (arrows). (E-H) Double heterozygous Dll1+/- Jag2+/- cochleae (E) demonstrate a similar increase in outer hair cells but not as large an increase in inner hair cells as Jag2-/- cochleae (F). Dll1+/- Jag2-/- (G) or Dll1hyp/-Jag2-/- (H) cochleae show a large increase in hair cell formation when compared with Jag2-/- cochleae (F). The hair cells in these cochleae are extremely disorganized. Scale bar: 50 µm.

View larger version (221K): [in a new window]   Fig. 2. Disorganization and loss of planar cell polarity in Dll1hyp/-Jag2-/- cochleae. Scanning electron microscopy of the basal region of cochleae of the indicated genotypes. (A,C,E) Low-power views. (B,D,F) Higher power views of the outer hair cell rows. Note the extremely disorganized and disoriented state of the hair cell bundles in the Dll1hyp/-Jag2-/- cochleae (F). In addition, some of the hair cells appear immature (arrow), with stereocilia covering the entire apical surface and not displaying the graded heights they would normally have acquired by this time. Some of this disorientation is also apparent in Jag2-/- cochleae (D), where some of the stereocila bundles on the outer hair cells do not orient properly (arrows). Scale bar: 50 µm in A,C,E; 10 µm in B,D,F.

View larger version (50K): [in a new window]   Fig. 3. Hair cells are dramatically increased in Dll1/Jag2 double mutant cochleae, whereas supporting cells are only modestly reduced. (A-D) Myosin VIIa (red), p27kip1 (green) and DAPI (blue nuclear stain) immunostained sections from E18.5 Dll1hyp/-Jag2-/- mutant and control cochleae. Supporting cells are clearly still present in Dll1hyp/-Jag2-/- mutant cochleae, as shown by the p27kip1-stained cells. However, some p27kip1-stained cells (presumably Deiter's cells) appeared to be missing in some sections (C,D, arrows). (E-H) E18.5 cochleae processed for in situ hybridization using the indicated probes. Expression of -tectorin or ß-tectorin in the various supporting cell populations did not appear to differ between controls and mutants, with the exception of ß-tectorin expression in Deiter's cells (H), which appears to be disorganized and reduced. (I) Hair and supporting cell counts from sections, as shown in A-D. Sections from three ears were counted for each group, either control or Dll1hyp/-Jag2-/-. Controls were either wild type, or Dll1 or Jag2 single heterozygotes. Counts of both hair cells and supporting cells were significantly different between Dll1hyp/-Jag2-/- mutant and control cochleae (*P<0.001, Student's t-test). However, the increase in hair cells did not equal the supporting cell losses (P<0.0001; one-way ANOVA). d, Deiter's cells; h, Hensen's cells; Ko, Koelliker's organ; iPh, inner phalangeal cells; p, pillar cells. Scale bars: in D, 50 µm for A-D; in H, 50 µm for E-H.

View larger version (97K): [in a new window]   Fig. 4. Continued proliferation occurs in Dll1hyp/-Jag2-/- mutant cochleae. (A-D) Immunohistochemistry on sections from E18.5 embryos was used to detect proliferating cells (green) in the organ of Corti after injections of BrdU from E14.5-E17.5. Hair cells are labeled with an antibody to detect myosin VIIa (red) and nuclei are stained with DAPI (blue). (A) Control section through the cochlea, demonstrating that there is normally no proliferation after E14.5 in the organ of Corti, although BrdU-labeled cells can been detected in Koelliker's organ and stromal cells beneath the organ of Corti. (B-D) Sections from Dll1hyp/-Jag2-/- cochleae, demonstrating examples of unexpected proliferating cells in the organ of Corti. The probable identities of these labeled cells based on the position of their nuclei are indicated. Some myosin VIIa-positive nuclei are located near the basement membrane in the position of supporting cell nuclei in the mutant cochleae (arrowheads in B and D). Asterisks indicate the tunnel of Corti in A-D. H, Hensen's cells; iPh, inner phalangeal cells; Ko, Koelliker's organ; oC, organ of Corti; OS, outer sulcus; pc, pillar cells; P/D, pillar/Deiter's cells. Scale bar: 50 µm.

View larger version (37K): [in a new window]   Fig. 5. Conditional Notch1 deletion in the ear causes supernumerary hair cell formation in the cochlea that resembles the Dll1hyp/-Jag2-/- mutant phenotype. (A,B) Lectin-stained whole-mount cochleae demonstrating the large increase in both inner and outer hair cells in Foxg1-Cre Notch1flox/- cochleae (B) when compared with Notch1flox/- cochleae (A), which appear normal. (C,D) Examples of the organ of Corti in sections from control (C) and Foxg1-Cre Notch1flox/- mutant (D) cochleae stained for myosin VIIa (hair cells, green) and p27kip1 (support cells, red). (E) Hair and supporting cell counts from 32 mid-modiolar sections, as shown in C and D (counts were done as for the Dll11/Jag2 double mutants). Sections from three ears were counted for each group, either control or Foxg1-Cre Notch1flox/-. Counts of both hair cells (HCs) and supporting cells (SCs) were significantly different between mutant and control cochleae (*P<0.001, **P<0.0001; Student's t-test). Similar to the Dll1/Jag2 double mutant cochleae, the increases in hair cells did not equal the supporting cell losses (P<0.0001; one-way ANOVA). Scale bars: in B, 100 µm for A,B; in D, 50 µm for C,D.

View larger version (41K): [in a new window]   Fig. 6. Model for the role of Notch signaling during hair and supporting cell differentiation in the cochlea. The diagram depicts on the left the signaling that takes place between precursor cells (Specification), and shows on the right the resulting cell fates (Differentiation) that are adopted as a result of this signaling in both wild-type (A) and Notch pathway mutant (B) cochleae. Red cells have reduced Notch activation and will ultimately differentiate as hair cells. The blue cells are those receiving sufficient Notch activation to prevent them from adopting the hair cell fate. The Notch-mediated inhibitory signal acts in two ways: (1) to inhibit the surrounding cells from adopting the hair cell fate; and (2) to suppress continued proliferation of the surrounding cells. In the Notch mutant cochleae precursor cells, the inhibitory signal is reduced. This leads to the production of supernumerary hair cells and the continued cell division of some precursor cells that then preferentially differentiate into supporting cells. HC, hair cell; SC, supporting cell.