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First published online 5 January 2005
doi: 10.1242/dev.01589

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Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation

Vertebrate Development Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (42K): [in a new window]   Fig. 1. Schematic diagram of development of the chick inner ear and its sensory patches. (A) The inner ear of a bird originates by invagination of the otic placode and remodelling of the resulting otic vesicle to form the labyrinth. The mature structure contains seven vestibular sensory patches, involved in perception of gravity and acceleration: three cristae (in the ampullae of the semicircular canals) and four maculae, those of the utricle, the saccule, the lagena, and the macula neglecta (which we neglect). The basilar papilla, an elongated sensory region extending along the cochlear duct, serves auditory function. (B) The patterns of Notch ligand expression in a sensory patch before, during and after hair-cell differentiation. One of the earliest genes to be expressed in prospective sensory patches codes for the Notch ligand Ser1. Within the Ser1 domains, nascent hair cells expressing Delta1 can be detected from E3.5 in vestibular regions, and from E5 in the cochlear duct. Delta1 expression foreshadows the differentiation of hair cells, which become identifiable by morphological and other molecular criteria about 24 hours later. Notch1 itself is expressed in sensory as well as non-sensory regions of the developing otocyst.

View larger version (142K): [in a new window]   Fig. 2. Expression of GFP in the chick inner ear after electroporation of control IRES-GFP plasmid DNA. (A) Low magnification view of the cochlear duct of an E9 chick embryo electroporated at stage HH 17 (E2.5). In the basilar papilla and the lagenar macula, each red dot corresponds to the stereociliary bundle of one hair cell. (B) High magnification view of a more proximal region of the BP of the same specimen. Scattered GFP-positive cells (green) include both hair cells (with rounded GFP-positive cell body and HCA-positive apical surface, arrowheads) and supporting cells (elongated cell bodies and absence of HCA staining). (C,D) Utricular macula of an E8 chick embryo electroporated at stage HH 13 (E2). Several groups of GFP-positive cells are present within this sensory patch. (D) Transverse view of this epithelium at higher magnification. Some of the GFP-positive cells are hair cells (asterisk), with HCA-positive hair bundles and HCS-1-positive cytoplasm; others are supporting cells (arrows), with elongated cell bodies extending below the hair cell layer. All images other than (D) are projections of confocal optical sections of immunostained whole mounts; (D) is a single optical section. Counterstain for actin (blue) in B and C, combined with the red HCA fluorescence, makes hair bundles appear purple. BP, basilar papilla; L, lagenar macula.

View larger version (84K): [in a new window]   Fig. 3. Notch activity within a sensory patch acts cell-autonomously to inhibit hair-cell differentiation. (A) Whole-mount immunostaining in the utricle of an E9 embryo electroporated at stage HH 17 (E2.5) with the NICD-IRES-GFP construct. In this specimen, GFP-positive cells are clustered and form a patch completely devoid of hair cells. Around the GFP-positive region, hair cells differentiate normally. (B) Whole-mount immunostaining in the saccule of an E9 embryo electroporated at stage HH 17 (E2.5) with NICD-IRES-GFP construct. In this specimen, GFP-positive cells are mixed with GFP-negative (untransfected) cells. Two different GFP-positive regions are outlined (1, 2). In both regions, hair cell density is diminished compared with adjacent regions. In region 2, however, some HCA-positive cells (hair cells) can be seen in the central part of the transfected patch. (C) Careful examination of region 2 at higher magnification, and in z-series of optical sections (Z1 to Z4, from the apical surface of the epithelium to deeper planes) reveals that these HCA positive cells are all GFP-negative (arrowheads); only the cells transfected with the NICD-IRES-GFP construct are inhibited from differentiating as hair cells.

View larger version (131K): [in a new window]   Fig. 4. Notch activity outside sensory patches can induce ectopic hair-cell differentiation. Whole-mount immunostaining in auditory and vestibular epithelia of E10 embryos electroporated at stage HH 13 (E2) with NICD-IRES-GFP. (A) At low magnification, the overall pattern of HCA staining is normal in the basilar papilla and lagenar macula (compare with Fig. 2A), but several patches of ectopic hair cells (arrows) have differentiated in the region inferior to the basilar papilla. (B) Higher magnification view of clusters of ectopic hair cells neighbouring the distalmost region of the basilar papilla (white box in A). The ectopic hair cells lie within GFP-positive groups of cells (arrowheads). (C) Low magnification view of sensory epithelium of a crista and the surrounding non-sensory tissues. At some distance from the crista, clusters of GFP-positive cells are present (white box). (D) Higher magnification reveals that some members of each cluster of GFP-positive cells have differentiated as ectopic hair cells (asterisks). The optical section here is transverse to the ectopic sensory epithelium, and the hair bundles (HCA-positive) are on the apical surface. (E) Two compact groups of GFP-positive cells forming spherical or vesicular aggregates. One of these structures contains hair cells (asterisk); the other does not. (F) An ectopic sensory-organ-like structure located at the proximal end of the cochlear duct. GFP-positive cells form a tubular protrusion (arrowheads) ending in a hollow epithelial pouch containing hair cells (asterisk), whose HCA-positive hair bundles face the central lumen. BP, basilar papilla; L, lagenar macula.

View larger version (118K): [in a new window]   Fig. 5. Notch activity outside sensory patches can induce ectopic expression of multiple sensory-patch markers. (A) Cryosections of the cochlear duct from an E7 embryo electroporated at stage HH 13 (E2) with NICD-IRES-GFP. In the cochlear duct at this stage, TuJ1 staining is a feature of sensory progenitors and nascent hair cells (Molea et al., 1999), as seen in the proximal (upper) part of the basilar papilla. A cluster of NICD-IRES-GFP-positive cells is present in the lateral wall of the cochlear duct (arrow), which is normally devoid of hair cells. At high magnification (right panel), the cells of this cluster are seen to express high levels of TuJ1. (B) Whole-mount in-situ hybridisation for Bmp4 (red) and immunostaining for GFP (green) in vestibular epithelia of E5 embryos electroporated at stage HH 13 (E2) with NICD-IRES-GFP. The crista expresses Bmp4 in the normal fashion for a sensory patch, while a group of GFP-positive cells express Bmp4 ectopically (arrowheads). (C) Cryosections of the dorsal region of the posterior crista from an E7 embryo electroporated at stage HH 13 (E2) with NICD-IRES-GFP. Some of the NICD-IRES-GFP-transfected cells have differentiated as ectopic hair cells, labelled for HCS-1 (arrows). These cells were also positive for HCA (not shown). (D,E) Immunostaining of GFP and ß-III tubulin (TuJ1) in transverse paraffin sections of the inner ear from an E9 embryo electroporated at stage HH 13 (E2) with NICD-IRES-GFP. Left and right panels in D show the electroporated and non-electroporated sides of the embryo, respectively; nerve cells and neurites penetrating sensory patches are strongly TuJ1-positive (dorsal is up). On the electroporated side, in addition to the utricle, saccule and cochlear duct/basilar papilla, an extra epithelial region with a central lumen can be seen (asterisk). At higher magnification (J), TuJ1-positive nerve fibres from the vestibular ganglion are seen to contact the GFP-positive cells in the ectopic end-organ-like structure. BP, basilar papilla; CG, cochlear ganglion; NT, neural tube; S, saccule; U, utricle; VG, vestibular ganglion.

View larger version (133K): [in a new window]   Fig. 6. Ectopic Ser1 expression in NICD-IRES-GFP transfected cells. Embryos electroporated at stage HH 13 (E2) with NICD-IRES-GFP construct. (A) Whole-mount immunostaining 48 hours post-electroporation. Ser1 is detected in its normal broad ventromedial domain of the otocyst, with a clear limit of expression (dashed line). It is also detected ectopically in NICD-IRES-GFP transfected cells (arrows). (B) Transverse cryosections 5 days post-electroporation. The saccular macula shows normal Ser1 expression, while ectopic Ser1 expression is seen in a cluster of GFP-positive transfected cells (arrow). S, saccular macula.

View larger version (125K): [in a new window]   Fig. 7. In-situ hybridisation analysis of GFP mRNA persistence after electroporation of chick inner ear at stage HH 13 (E2) with HA-NICD-IRES-GFP construct. Representative micrographs of whole-mount specimens analysed at 24 hours, 3 days and 7 days post-electroporation. The number of cells expressing GFP mRNA decreases over time, and by 7 days post-electroporation scarcely any such cells are visible. The two cells positive for GFP mRNA in the 7-day specimen (shown enlarged in the insert) might be ones in which the plasmid has survived by integration into the host cell genome.