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First published online 22 February 2006
doi: 10.1242/dev.02284

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Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

¹ Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
² Hereditary Hearing Group, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, CB10 1SA, UK.
³ Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.

View larger version (158K): [in a new window]   Fig. 1. Loss of Jag1 antibody staining in the inner ear of Jag1 conditional knockout mice. (A,B) Confocal images of sagittal cryosections at E10.5 (dorsal to the left). (A) Littermate control. The Jag1 antibody stains a patch of cells (arrow) in the ventral part of the otocyst. (B) Jag1 conditional knockout. The patch of Jag1 staining is lost. (C,D) Confocal optical sections of apices of whole-mount E17.5 cochleas. (C) Littermate control. Jag1 is detected in a narrow band of cells (arrow) - the future sensory patch. (D) Jag1 conditional knockout. The Jag1 staining is lost.

View larger version (92K): [in a new window]   Fig. 2. Defective sensory patch development in the Jag1 conditional knockout cochlea. Confocal images of whole-mount E17.5 cochleas stained with fluorescent phalloidin. (A) In the apex of the control cochlea, faint lines of actin staining prefigure the appearance of the inner hair cells (arrow); no hint of outer hair cells is yet visible. (B) In the Jag1 conditional knockout littermate, the appearance of the cochlear apex is similar, with no sign of premature differentiation. (C) In the middle part of the control cochlea, the standard pattern of one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) is seen. (D) In the middle part of the Jag1 conditional knockout cochlea, approximately two disorganized rows of hair cells are produced. (E) In the mid-basal region of the Jag1 conditional knockout cochlea, hair cells lie in islands (arrows) separated by gaps of bare epithelium; the mid-basal region of the control cochlea (not shown) has the same pattern of four rows of hair cells as in the middle region (C).

View larger version (115K): [in a new window]   Fig. 3. The hair cells in the cochlea of the Jag1 conditional knockout have characteristics of inner hair cells. Confocal optical sections of whole mounts of the middle region of the cochlea at E17.5 are shown. (A) Control, stained with calretinin antibody. At this stage the inner hair cells (arrow) but not outer hair cells are calretinin-positive, and so are occasional inner pillar cells (arrowheads). (B) Jag1 conditional knockout cochlea. All the hair cells produced are stained for calretinin, as well as some adjacent cells (possibly inner pillar cells). (C) Control cochlea, stained with the nuclear dye DAPI and with phalloidin. A row of inner pillar cells with characteristic rectangular profiles can be seen in the wild type between the rows of inner (IHC) and outer hair cells (OHC). (D) In the Jag1 conditional knockout, cells with this morphology can be seen outside the disorganised rows of hair cells. The cells appear larger than in C merely because the plane of section is slightly different.

View larger version (87K): [in a new window]   Fig. 4. The vestibular apparatus is defective in the Jag1 conditional knockout. Specimens at E17.5. (A,B) Freshly dissected inner ears under the dissecting microscope; medial view, anterior towards the left. The Jag1 conditional knockout (B) has a truncated anterior semicircular canal (left, asterisk) and a missing posterior semicircular canal (right, asterisk). (C,D) Confocal optical sections through the horizontal crista and utricular macula, stained for actin and DNA. In the wild-type control (C), nuclei of hair cells and supporting cells are arranged in two distinct layers (arrows in C and C'); in the Jag1 conditional knockout (D), the corresponding patches are smaller and there is only one layer of nuclei, reflecting absence of hair cells. (C',D') Enlargements of the horizontal crista. (E,F) Confocal optical sections of the saccular macula, stained for actin, DNA and calretinin. The mutant macula appears to be unaffected by loss of Jag1.

View larger version (68K): [in a new window]   Fig. 5. Hair cells are produced early and in excess in the Delta1 conditional knockout cochlea. Confocal optical sections of whole mounts of cochleas at E17.5. (A) In the apex of the wild-type mouse cochlea, stained with phalloidin, no hair cells are visible as yet. (B,B') In the apex of the cochlea of a Delta1 conditional knockout littermate, hair cells are already plainly visible and present in excess (arrow; B' shows detail). (C) Middle region of wild-type cochlea shows a narrow band of Jag1 staining, corresponding to the future sensory patch. (D) Delta1 conditional knockout shows a wider band of Jag1 staining, corresponding to the enlarged and precocious sensory patch, and suggesting an increase in the numbers of supporting cells (which are Jag1-positive) as well as hair cells (which are not). (E,F) A milder excess of hair cells is seen in the middle and basal parts of the Delta1 conditional knockout cochlea (F), with one extra row of outer hair cells and many more inner hair cells than in the littermate control (E). (G,H) Confocal optical sections of the middle region of the cochlea in a plane passing through the bodies of the supporting cells at a deeper level than the hair cells. The membranes of the supporting cells are stained with Jag1 antibody, and the mutant has slightly more of these cells per unit length of cochlea.

View larger version (131K): [in a new window]   Fig. 6. Delta1 conditional knockout mice have vestibular defects. Coronal cryosections of vestibular regions of the inner ear of mutants and littermate controls stained with Jag1 antibody and fluorescent phalloidin. (A,B) Saccule. The saccular macula is much smaller (and sometimes missing) in the Delta1 conditional knockout (B), when compared with the Foxg1-Cre littermate control (A). (C,D) Utricle and horizontal crista. The utricular macula is also drastically reduced in the mutant (D) compared with the control (C), though the crista is less affected.

View larger version (86K): [in a new window]   Fig. 7. p27Kip1 expression is drastically reduced in the Jag1 but not the Delta1 conditional knockout cochlea. Confocal optical sections of whole-mount E14.5 cochleas stained with anti-p27Kip1 antibody and fluorescent phalloidin. The bright staining (asterisks) in tissue outside the sensory epithelium in the apex of the cochlea is non-specific and occurs in an area damaged during dissection. (A) Wild-type control. p27Kip1 is expressed in a band of cells (arrow) corresponding to the future organ of Corti. (B) Delta1 conditional knockout littermate. There is little change in the expression of p27Kip1 in the developing sensory patch, except that it extends further towards the apex in accordance with the premature differentiation of this region. (C) Wild-type littermate control for the Jag1 conditional knockout. (D) Jag1 conditional knockout, showing complete loss of expression of p27Kip1 in the developing sensory patch (arrows in C,D). (E) Another Jag1 conditional knockout specimen, showing some weak residual expression of p27Kip1 (arrow).